ES2961686T3 - Modulation of apolipoprotein C-III (APOCIII) expression in lipoprotein lipase deficient (LPLD) populations - Google Patents

Abstract

Provided herein are methods, compounds and compositions for reducing the expression of ApoCIII mRNA and protein in a patient with Fredrickson dyslipidemia Type I, FCS, LPLD. Also provided herein are methods, compounds and compositions for treating, preventing, delaying or ameliorating Fredrickson's dyslipidemia type I, FCS, LPLD, in a patient. Additionally provided herein are methods, compounds and compositions for increasing HDL levels and/or improving the ratio of TG to HDL and reducing plasma lipids and plasma glucose in a patient with Fredrickson Type I dyslipidemia, FCS, LPLD. Such methods, compounds and compositions are useful for treating, preventing, delaying or ameliorating any one or more of pancreatitis, cardiovascular diseases or metabolic disorders, or one of their symptoms. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Modulation of apolipoprotein C-III (APOCIII) expression in lipoprotein lipase deficient (LPLD) populations

Field of Invention

Disclosed herein are methods, compounds and compositions for reducing the expression of Apolipoprotein C-III (ApoCIII) mRNA and protein, reducing triglyceride levels and increasing high-density lipoprotein (HDL) levels or HDL activity in patients with Fredrickson's dyslipidemia Type I. In addition, compounds and compositions are also disclosed herein for use in the treatment of Fredrickson's dyslipidemia Type I or associated disorders thereof.

Background

Lipoproteins are globular micelle-like particles consisting of a nonpolar core of acylglycerols and cholesteryl esters surrounded by an amphiphilic coating of protein, phospholipid, and cholesterol. Lipoproteins have been classified into five broad categories based on their functional and physical properties: chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins ( HDL). Chylomicrons transport dietary lipids from the intestine to the tissues. VLDL, IDL and LDL transport triacylglycerols and cholesterol from the liver to the tissues. HDL transports endogenous cholesterol from tissues to the liver.

Apolipoprotein ApoC-III (also called APOC3, APOC-III, ApoCIII and APO C-III) is a constituent of HDL and triglyceride-rich (TG) lipoproteins. Elevated ApoCIII is associated with elevated TG levels and diseases such as cardiovascular disease, metabolic syndrome, obesity and diabetes (Chan et al., Int J Clin Pract, 2008, 62:799-809; Onat et at., Atherosclerosis, 2003, 168:81-89; Mendivil et al., Circulation, 2011, 124:2065-2072; 278-283; Ooi et al., Clin. Sci, 2008. 114: 611-624;

102: 1886-1892; Lee et al., Arterioscler Thromb Vasc Biol, 2003. 23: 853-858). ApoCIII slows the clearance of TG-rich lipoproteins by inhibiting lipolysis, both through inhibition of lipoprotein lipase (LPL) and by interfering with the binding of lipoproteins to the cell surface glycosaminoglycan matrix (Shachter, Curr. Opin. Lipidol, 2001, 12, 297-304). As ApoCIII inhibits LPL leading to a decrease in TG lipolysis, it would be unexpected for ApoCIII inhibition to have a beneficial effect in LPL-deficient (LPLD) subjects.

LPLD is characterized by the inability of affected individuals to produce functionally active LPL. LPL is mainly produced in skeletal muscle, adipose tissue, and cardiac muscle and has multiple key functions, including catabolism of TG-rich lipoproteins (e.g., VLDL) and chylomicrons (CM). TG unloading of CM (and VLDL) normally protects against excessive postprandial increase in CM and TG mass. In LPLD, the<l>P<l>is dysfunctional and more than 12 hours after meals hyperTG and chylomicroemia are still present and visible as lipemia.

The Fredrickson system is used to classify the primary (genetic) causes of dyslipidemia, such as hypertriglyceridemia in patients. Fredrickson Type I (also known as LPLD or Familial Chylomicronemia Syndrome (FCS)) is usually caused by mutations of either the LPL gene or the ApoC-II cofactor of the gene, resulting in the inability of affected individuals to produce LPL. functionally active (i.e. LPLD). Patients have mutations that are either homozygous (having the same mutation on each allele) or compound heterozygous (having different mutations on each allele). The prevalence is approximately 1 in 1,000,000 in the general population and much higher in South Africa and Eastern Quebec as a result of a founder effect.

Currently, patients with Fredrickson Type I, FCS, LPLD, respond minimally, or not at all, to TG-lowering drugs such as statins, fibrates, and nicotinic acid (Tremblay et al., J Clin Lipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010, 20:742-747). The clinical management of patients with Fredrickson Type I, FCS, LPLD, generally consists of a severe reduction of all dietary fats to much less than 20% of caloric intake and the use of medium-chain TGs, which are absorbed through the portal system and therefore do not directly enter the plasma. Such a lifelong dietary regimen presents significant compliance problems for patients. Even when patients adhere to the diet and are strictly followed in a lipid clinic by a dietician and medical team, TGs often do not decrease below the threshold of increased pancreatitis risk. Recently, a gene therapy product (GlyberaR) has been approved in Europe for the treatment of adult LPLD patients who suffer from severe or multiple attacks of pancreatitis despite dietary fat restrictions. Patients treated with GlyberaR require administration of an immunosuppressive drug before and after treatment with GlyberaR. GlyberaR will only be offered through dedicated centers experienced in the treatment of LPLD and by specially trained doctors to ensure the continued safety of the treatment (http://www.uniqure.com/products/glybera/).

Therefore, there is still a need to provide patients with Fredrickson's dyslipidemia Type I, FCS, LPLD, new treatment options. Antisense technology is emerging as an effective means of reducing the expression of certain gene products and may prove to be exceptionally useful in a number of therapeutic, diagnostic and research applications for ApoCIII modulation. We have previously disclosed compositions and methods for inhibiting ApoCIII by antisense compounds in US 20040208856 (US Patent 7,598,227), US 20060264395 (US Patent 7,750,141), WO 2004/093783 and WO 2012/149495 . An antisense oligonucleotide targeting ApoCIII has been tested in a Phase I clinical trial and was shown to be safe. Currently, an antisense oligonucleotide targeting ApoCIII is in Phase II clinical trials to evaluate its efficacy in the treatment of diabetes or hypertriglyceridemia.

WO2011/123401 considers the use of a DGAT1 inhibitor for the treatment of hyperlipidemia diseases with chylomicronemia.

Summary of the Invention

The invention provides a specific inhibitor of ApoCIII for use in a method of i) treating, preventing, delaying or ameliorating LPLD or FCS in an animal; or ii) prevent, delay or ameliorate pancreatitis, or symptom thereof in an animal with LPLD or FCS, wherein the specific ApoCIII inhibitor comprises an antisense compound targeting ApoCIII, wherein the antisense compound is an interfering DNA compound .

Disclosure Summary

Disclosed herein is a method for treating, preventing, delaying or ameliorating Fredrickson's dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal. Also disclosed herein is a specific ApoCIII inhibitor for use in the treatment, prevention, delay or amelioration of Fredrickson dyslipidemia type I, FCS, LPLD.

Disclosed herein is a method of reducing triglyceride levels in an animal with Fredrickson Dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal.

Disclosed herein is a method of increasing HDL levels and/or improving the TG to HDL ratio in an animal with Fredrickson dyslipidemia type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal.

Disclosed herein is a method of preventing, delaying or ameliorating a disease, disorder, cardiovascular and/or metabolic condition, or symptom thereof, in an animal with Fredrickson's Dyslipidemia Type I, FCS, LPLD, comprising administering an amount therapeutically effective of a compound comprising a specific inhibitor of ApoCIII to the animal.

Disclosed herein is a method of preventing, delaying or ameliorating pancreatitis, or symptoms thereof, in an animal with Fredrickson's dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising an inhibitor. ApoCIII specific to the animal.

The specific ApoCIII inhibitor is an antisense compound, wherein the antisense compound is an interfering DNA compound.

In certain embodiments, the specific ApoCIII inhibitor is capable of inhibiting ApoCIII expression. Detailed description

It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. Use of the singular herein includes the plural unless specifically indicated otherwise. As used herein, use of “or” means “and/or” unless otherwise indicated. Furthermore, the use of the term "including", as well as other forms such as "includes" and "including", is not limiting. Furthermore, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising more than one subunit, unless specifically indicated otherwise.

The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

Definitions

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis.

Unless otherwise indicated, the following terms have the following meanings:

"2'-O-methoxyethyl" (also 2'-MOE, 2'-O(CH2)2-OCH3 and 2'-O-(2-methoxyethyl)) refers to a modification of O-methoxy-ethyl of the 2' position of a furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

"2'-O-methoxyethyl nucleotide" means a nucleotide comprising a modified sugar moiety of 2'-O-methoxyethyl.

"3' target site" refers to the nucleotide of a target nucleic acid that is complementary to the 3' most nucleotide of a particular antisense compound.

"Target site 5" refers to the nucleotide of a target nucleic acid that is complementary to the most 5' nucleotide of a particular antisense compound.

"5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. A 5-methylcytosine is a modified nucleobase.

"Approximately" means within ±10% of a value. For example, if it is stated, "a marker can be increased by approximately 50%, it is implied that the marker can be increased by 45%-55%.

"Active pharmaceutical agent" means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, an antisense oligonucleotide targeting ApoCIII is an active pharmaceutical agent.

"Active target region" or "target region" means a region targeted by one or more active antisense compounds. "Active antisense compounds" means antisense compounds that reduce target nucleic acid levels or protein levels.

"Administered concomitantly" refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifested in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents do not need to manifest at the same time. The effects only need to overlap over a period of time and do not need to be coextensive.

"Administer" means providing a pharmaceutical agent to an individual, and includes, but is not limited to, administering by a medical professional and self-administration.

"Agent" means an active substance that can provide a therapeutic benefit when administered to an animal. "First agent" means a therapeutic compound of the invention. For example, a first agent may be an antisense oligonucleotide targeting ApoCIII. "Second agent" means a second therapeutic compound of the invention (for example, a second antisense oligonucleotide that targets ApoCIII) and/or a non-ApoCIII therapeutic compound.

"Improvement" means a decrease in at least one indicator, sign or symptom of an associated disease, disorder or condition. The severity of the indicators can be determined by subjective or objective measures, which are known to those skilled in the art.

"Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

"Antisense activity" means any detectable or measurable activity attributable to hybridization of an antisense compound with its target nucleic acid. In certain embodiments, the antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by said target nucleic acid.

"Antisense compound" means an oligomeric compound that is capable of undergoing hybridization with a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as antisense oligonucleotides, siRNA, shRNA, ssRNA, and occupancy-based compounds.

"Antisense inhibition" means the reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

"Antisense oligonucleotide" means a single-stranded oligonucleotide that has a nucleobase sequence that allows hybridization with a corresponding region or segment of a target nucleic acid. As used herein, the term "antisense oligonucleotide" encompasses pharmaceutically acceptable derivatives of the compounds described herein.

"ApoA5", "Apolipoprotein AV" or "ApoA-V" means any nucleic acid or protein sequence that encodes ApoA5.

"ApoCII", "Apolipoprotein C-II" or "ApoC2" means any nucleic acid or protein sequence that encodes ApoCII. The ApoCII protein is a component of chylomicron particles and VLDL and activates LPL to hydrolyze TG.

"ApoCIII", "Apolipoprotein C-III" or "ApoC3" means any nucleic acid or protein sequence that encodes ApoCIII. For example, in certain embodiments, an ApoCIII includes a DNA sequence that encodes ApoCIII, an RNA sequence transcribed from DNA that encodes ApoCIII (including genomic DNA comprising introns and exons), an mRNA sequence that encodes ApoCIII, or a sequence peptide that encodes ApoCIII.

"Specific ApoCIII inhibitor" refers to any agent capable of specifically inhibiting ApoCIII mRNA expression and/or ApoCIII protein expression or activity at the molecular level. For example, specific inhibitors of ApoCIII include nucleic acids (including antisense compounds), peptides, antibodies, small molecules and other agents capable of inhibiting the expression of ApoCIII mRNA and/or ApoCIII protein. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is an oligonucleotide targeting ApoCIII. In certain embodiments, the ApoCIII-targeting oligonucleotide is a modified ApoCIII-targeting oligonucleotide. In certain embodiments, the ApoCIII-targeting oligonucleotide has a sequence as shown in SEQ ID NO: 3 or another sequence, for example, such as those disclosed in US Patent 7,598,227, US Patent 7,750,141 , PCT publication WO 2004/093783 or WO 2012/149495. In certain embodiments, by specifically modulating the ApoCIII mRNA level and/or ApoCIII protein expression, specific ApoCIII inhibitors can affect components of the lipogenic pathway. Similarly, in certain embodiments, specific ApoCIII inhibitors may affect other molecular processes in an animal.

"ApoCIII mRNA" means an mRNA that encodes an ApoCIII protein.

"ApoCIII protein" means any protein sequence that encodes ApoCIII.

"Atherosclerosis" means a hardening of the arteries that affects the large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called "atheromas" or "plaques," which consist mainly of cholesterol and other fats, calcium, and scar tissue, and damage the lining of the arteries.

"Bicyclic sugar" means a furosyl ring modified by the bridging of two non-germ ring atoms. A bicyclic sugar is a modified sugar.

"Bicyclic nucleic acid" or "BNA" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms in the furanose ring, thereby forming a ring system. bicyclic.

"Cap structure" or "end cap moiety" means chemical modifications, which have been incorporated into either of the terminal ends of an antisense compound.

"Cardiovascular disease" or "cardiovascular disorder" refers to a group of conditions related to the heart, blood vessels, or circulation. Examples of cardiovascular diseases include, but are not limited to, aneurysm, angina pectoris, arrhythmia, atherosclerosis, cerebrovascular disease (stroke), coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia, and hypercholesterolemia.

"Chemically distinct region" refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2-O-methoxyethyl modifications.

"Chimeric antisense compound" means an antisense compound that has at least two chemically distinct regions.

"Cholesterol" is a sterol molecule found in the cell membranes of all animal tissues. Cholesterol must be transported in an animal's blood plasma by lipoproteins that include very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). "Plasma cholesterol" refers to the sum of the esterified and/or non-esterified cholesterol of all lipoproteins (VDL, IDL, LDL, HDL) present in the plasma or serum.

"Cholesterol absorption inhibitor" means an agent that inhibits the absorption of exogenous cholesterol obtained from the diet.

"Co-management" means the management of two or more agents to an individual. The two or more agents may be in a single pharmaceutical composition, or they may be in separate pharmaceutical compositions. Each of the two or more agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

"Complementarity" means the ability for nucleobase pairing of a first nucleic acid and a second nucleic acid. In certain embodiments, the complementarity between the first and second nucleic acids may be between two DNA strands, between two RNA strands, or between a DNA strand and an RNA strand. In certain embodiments, some of the nucleobases on one strand match a complementary hydrogen bonding base on the other strand. In certain embodiments, all nucleobases on one strand match a complementary hydrogen bonding base on the other strand. In certain embodiments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid. In certain cases, an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.

"Contiguous nucleobases" means nucleobases immediately adjacent to each other.

"Restrained ethyl" or "cEt" refers to a bicyclic nucleoside having a furanosyl sugar comprising a methyl(methyleneoxy)(4'-CH(CH3)-O-2') bridge between the 4' carbon atoms. and 2'.

"Cross-reactive" means an oligomeric compound targeting one nucleic acid sequence that can hybridize to a different nucleic acid sequence. For example, in some cases, an antisense oligonucleotide targeting human ApoCIII can cross-react with a murine ApoCIII. Whether an oligomeric compound cross-reacts with a nucleic acid sequence other than its designated target depends on the degree of complementarity the compound has with the non-target nucleic acid sequence. The greater the complementarity between the oligomeric compound and the non-target nucleic acid, the more likely it is that the oligomeric compound will cross-react with the nucleic acid.

"Cure" means a method that restores health or a prescribed treatment for a disease.

"Coronary heart disease (CHD)" means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

"Deoxyribonucleotide" means a nucleotide that has a hydrogen at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides can be modified with any of a variety of substituents.

"Diabetes mellitus" or "diabetes" is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) as a result of insufficient insulin levels or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) trying to compensate for increased urination, blurred vision due to the effects of high blood glucose on the optics of the eye, unexplained weight loss, and lethargy.

"Diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia" means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides, and elevated small, dense LDL particles.

"Diluent" means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition may be a liquid, for example, saline.

"Dyslipidemia" refers to a disorder of lipid and/or lipoprotein metabolism, including overproduction or deficiency of lipids and/or lipoproteins. Dyslipidemias can be manifested by elevation of lipids such as chylomicron, cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein cholesterol (LDL). An example of a dyslipidemia is chylomicronemia.

"Dosage unit" means a form in which a pharmaceutical agent is provided, for example, pill, tablet or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing a lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing a reconstituted antisense oligonucleotide.

"Dose" means a specified amount of a pharmaceutical agent provided in a single administration, or over a specified period of time. In certain embodiments, a dose may be administered in one, two or more boluses, tablets or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be set as the amount of pharmaceutical agent per hour, day, week, or month. Doses can also be expressed as mg/kg or g/kg.

"Effective amount" or "therapeutically effective amount" means the amount of active pharmaceutical agent sufficient to effect a desired physiological result in an individual in need of the agent. The effective amount may vary between individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, the evaluation of the medical condition of the individual and other relevant factors.

"Fibrates" are agonists of the peroxisome proliferator-activated receptor w (PPAR-a), which act through transcription factors that regulate several steps in lipid and lipoprotein metabolism. By interacting with PPAR-a, fibrates recruit different cofactors and regulate gene expression. As a consequence, fibrates are effective in reducing fasting TG levels as well as remaining postprandial TG and TRL particles. Fibrates also have modest LDL-C-lowering and HDL-C-elevating effects. The reduction in the expression and levels of ApoC-III is a constant effect of PPAR-α agonists (Hertz et al. J Biol Chem, 1995, 270(22):13470-13475). A 36% reduction in plasma ApoC-III levels was reported with fenofibrate treatment in the metabolic syndrome (Watts et al. Diabetes, 2003, 52:803-811). However, fibrates have been ineffective in treating LPLD subjects with hypertriglyceridemia.

The "Fredrickson" system is used to classify the primary (genetic) causes of dyslipidemia into various subgroups or types. Types of dyslipidemia that may be amenable to therapy with the compounds disclosed herein include, but are not limited to, Fredrickson Type I, FCS, LPLD.

"Fredrickson Type I" is also known as "lipoprotein lipase deficiency", "LPLD", "Familial Chylomicronemia Syndrome" or "FCS" and exists in several forms: Type 1a (also known as Buerger-Gruestz syndrome ) is a lipoprotein lipase deficiency commonly due to LPL deficiency or altered apoC-II; Type Ib (also known as familial apoprotein CII deficiency) is a condition caused by a lack of the lipoprotein lipase activator apoprotein C-II; and Type Ic is a chylomicronemia due to circulating lipoprotein lipase inhibitor. Type I is a rare disorder that usually presents in childhood. It is characterized by severe elevations in chylomicrons and extremely high TG levels (always far exceeding 1000 mg/dl and not infrequently increasing as high as 10,000 mg/dl or more) with episodes of abdominal pain, recurrent acute pancreatitis, xanthomas. eruptive skin lesions, and hepatosplenomegaly. Patients rarely develop atherosclerosis, perhaps because their plasma lipoprotein particles are too large to enter the arterial intima (Nordestgaard et al., J Lipid Res, 1988, 29:1491-1500; Nordestgaard et al., Arteriosclerosis , 1988, 8:421-428). Type I is usually caused by mutations of either the LPL gene or the gene's apoC-II cofactor, resulting in the inability of affected individuals to produce sufficient functionally active LPL. Patients are either homozygous for such mutations or compound heterozygotes. Fredrickson Type I may also be due to mutations in GPIHBP1, APOA5, LMF1, or other genes that lead to dysfunctional LPL Brunzell, In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993 2013.October 12, 1999 [updated December 15, 2011]. Additionally, Fredrickson Type I, in some cases, may be due to the presence of LPL inhibitors (e.g., anti-LPL antibodies) in an individual causing dysfunctional LPL. The prevalence of Fredrickson Type I is approximately 1 in 1,000,000 in the general population and much higher in South Africa and Eastern Quebec as a result of a founder effect. Patients respond minimally, or not at all, to drugs that lower TG (Tremblay et al., J Clin Lipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010, 20:742-747 ), and therefore dietary fat restriction to 20 grams/day or less is used to control the symptoms of this rare disorder.

"Fredrickson Type II" is the most common form of primary hyperlipidemia. It is further classified into Type IIa and Type IIb, depending mainly on whether there is elevation of VLDL in addition to LDL cholesterol (LDL-C). Type IIa (familial hypercholesterolemia) may be sporadic (due to dietary factors), polygenic, or truly familial as a result of a mutation in either the LDL receptor gene on chromosome 19 (0.2% of the population) or the of apolipoprotein B (apoB) (0.2%). The familial form is characterized by tendon xanthoma, xanthelasma, and premature cardiovascular disease. The incidence of this disease is approximately 1 in 500 for heterozygotes and 1 in 1,000,000 for homozygotes. Type IIb (also known as familial combined hyperlipoproteinemia) is a mixed hyperlipidemia (cholesterol and high TG levels), caused by elevations in LDL-C and VLDL. High VLDL levels are due to overproduction of substrates, including TG, acetyl CoA, and an increase in B-100 synthesis. They can also be caused by decreased LDL clearance. The prevalence in the population is approximately 10%.

"Fredrickson Type III" (also known as dysbetalipoproteinemia) is a remnant elimination disease, or broad beta disease (Fern et al., J Clin Pathol, 2008, 61: 1174-118). It is due to cholesterol-rich VLDL (p-VLDL). Typically, patients with this condition have elevated plasma cholesterol and TG levels due to impaired clearance of chylomicron and VLDL remnants (e.g., IDL). Impaired clearance is due to a defect in apolipoprotein E (apoE). Normally functioning apoE contained in the remnants would allow binding to the LDL receptor and clearance from circulation. Accumulation of remnants in affected individuals can result in xanthomatosis and premature coronary and/or peripheral vascular disease. The most common cause for Type III is the presence of the apoE E2/E2 genotype. Its prevalence has been estimated at approximately 1 in 10,000.

"Fredrickson Type IV" (also known as familial hypertriglyceridemia) is an autosomal dominant condition that occurs in approximately 1% of the population. TG levels are elevated as a result of excess hepatic VLDL production or heterozygous LPL deficiency, but are almost always less than 1000 mg/dL. Serum cholesterol levels are usually within normal limits. The disorder is heterogeneous and the phenotype is strongly influenced by environmental factors, particularly carbohydrate and ethanol consumption.

"Fredrickson Type V" has high VLDL and chylomicrons. It is characterized by carriers of loss-of-function LPL gene variants associated with LPL activity of at least 20% (i.e., partial LPL deficiency compared to Fredrickson type I). These patients present with lactescent plasma and severe hypertriglyceridemia due to chylomicrons and VLDL. TG levels are invariably greater than 1000 mg/dl and total cholesterol levels are always elevated. The LDL-C level is usually low. It is also associated with an increased risk of acute pancreatitis, glucose intolerance, and hyperuricemia. Symptoms usually present in adulthood (>35 years) and, although the prevalence is relatively rare, it is much more common than patients with homozygous or compound heterozygous LPL deficiency.

"Completely complementary" or "100% complementary" means that each nucleobase in a nucleobase sequence of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid.

"Gapmer" means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically other than the nucleoside or nucleosides that comprise the outer regions. The inner region may be called the "gap" or "gap segment" and the outer regions may be called "wings" or "wing segments."

"Gap-extended" means a chimeric antisense compound having a gap segment of 12 or more contiguous 2-deoxyribonucleosides positioned between and immediately adjacent to the 5' and 3' wing segments having one to six nucleosides.

"Genetic testing" means examining genotypic variations or mutations in an animal. In some cases, the mutation can lead to a phenotypic change in the animal. In certain cases, the phenotypic change is, or leads to, a disease, disorder or condition in the animal. For example, mutations in the LPL or ApoC-II genes can lead to Fredrickson dyslipidemia Type I, FCS, LPLD. Genetic screening can be performed by any of the techniques known in the art, for example, sequencing the LPL or ApoC-II gene or mRNA to detect mutations. The sequence from the animal being examined is compared to the sequence from a normal animal to determine if there are any mutations in the sequence. Alternatively, for example, identification of mutations in the LPL or ApoC-II gene or mRNA can be performed using PCR amplification and gel or chip analysis.

"Glucose" is a monosaccharide used by cells as an energy source and inflammatory intermediate. "Plasma glucose" refers to the glucose present in plasma.

"High-density lipoprotein" or "HDL" refers to a macromolecular complex of lipids (cholesterol, triglycerides and phospholipids) and proteins (apolipoproteins (apo) and enzymes). The surface of HDL contains mainly apolipoproteins A, C, and E. The function of some of these apoproteins is to direct HDL from peripheral tissues to the liver. Serum HDL levels can be affected by underlying genetic causes (Weissglas-Volkov and Pajukanta, J Lipid Res, 2010, 51:2032-2057). Epidemiological studies have indicated that increased levels of HDL protect against cardiovascular disease or coronary heart disease (Gordon et al., Am. J. Med. 1977. 62: 707-714). These effects of HDL are independent of triglyceride and LDL concentrations. In clinical practice, low plasma HDL is more commonly associated with other disorders that increase plasma triglycerides, for example, central obesity, insulin resistance, type 2 diabetes mellitus, and kidney disease (chronic renal failure or nephritic proteinuria). (Kashyap. Am. J. Cardiol. 1998. 82: 42U-48U).

"High Density Lipoprotein-Cholesterol" or "HDL-C" means cholesterol associated with high-density lipoprotein particles. HDL-C concentration in serum (or plasma) is typically quantified in mg/dl or nmol/l. "HDL-C" and "plasma HDL-C" mean serum and plasma HDL-C, respectively.

"HMG-CoA reductase inhibitor" means an agent that acts through the inhibition of the HMG-CoA reductase enzyme, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin and simvastatin.

"Hybridization" means the pairing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

"Hypercholesterolemia" means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL cholesterol, and VLDL cholesterol, according to the guidelines of the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med. (1988) 148, 36-39).

"Hyperlipidemia" or "hyperlipidemia" is a condition characterized by elevated levels of serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in circulating blood are cholesterol, low-density lipoproteins, very low-density lipoproteins, chylomicrons, and triglycerides. The Fredrickson classification of hyperlipidemias is based on the pattern of TG and cholesterol-rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation, and is commonly used to characterize the major causes of hyperlipidemias such as hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965, 31:321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1): 34-42).

"Hypertriglyceridemia" means a condition characterized by elevated triglyceride levels. Hypertriglyceridemia is the consequence of increased production and/or reduced or delayed catabolism of lipoproteins rich in triglycerides (TG): VLDL and, to a lesser extent, chylomicrons (CM). Its etiology includes primary factors (i.e., genetic causes) and secondary factors (other underlying causes such as diabetes, metabolic syndrome/insulin resistance, obesity, physical inactivity, smoking, excess alcohol, and a very high carbohydrate diet) or, more often a combination of both (Yuan et al. CMAJ, 2007, 176:1113-1120). Hypertriglyceridemia is a common clinical feature associated with an increased risk of cardiometabolic disease (Hegele et al. 2009, Hum Mol Genet, 18: 4189-4194; Hegele and Pollex 2009, Mol Cell Biochem, 326: 35-43) as well as appearance of acute pancreatitis in the most severe forms (Toskes 1990, Gastroenterol Clin North Am, 19: 783 791; Gaudet et al. 2010, Atherosclerosis Supplements, 11: 55-60; Catapano et al. 2011, Atherosclerosis, 217S: S1- S44; Tremblay et al. 2011, J Clin Lipidol, 5: 37-44). Examples of cardiometabolic disease include, but are not limited to, diabetes, metabolic syndrome/insulin resistance, and genetic disorders such as familial chylomicronemia syndrome (FCS), familial combined hyperlipidemia, and familial hypertriglyceridemia. Borderline high TG levels (150-199 mg/dl) are commonly found in the general population and are a common component of metabolic syndrome/insulin resistance states. The same is true for high TG levels (200,499 mg/dl), except that as plasma TG levels increase, underlying genetic factors play an increasingly important etiologic role. Very high TG levels (>500 mg/dl) are also more frequently associated with elevated MC levels, and are accompanied by an increased risk of acute pancreatitis. The risk of pancreatitis is considered clinically significant if TG levels exceed 880 mg/dl (>10 mmol) and the 2011 European Atherosclerosis Society/European Society of Cardiology (EAS/ESC) guidelines state that actions to prevent acute pancreatitis are mandatory (Catapano et al. 2011, Atherosclerosis, 217S: S1-S44). According to the 2011 EAS/ESC guidelines, hypertriglyceridemia is the cause of approximately 10% of all cases of pancreatitis, and the development of pancreatitis can occur at TG levels between 440-880 mg/dl . Based on evidence from clinical studies demonstrating that elevated TG levels are an independent risk factor for atherosclerotic CVD, guidelines from both the National Cholesterol Education Program Adult Treatment Panel III (NCEP 2002, Circulation, 106: 3143-421) and the American Diabetes Association (ADA 2008, Diabetes Care, 31: S12-S54.) recommended a target TG level of less than 150 mg/dl to reduce cardiovascular risk.

"identifying" or "diagnosing" an animal with an identified disease, disorder or condition means identifying, by methods known in the art, a subject prone to, or having, the identified disease, disorder or condition.

"Identifying" or "diagnosing" an animal with Fredrickson dyslipidemia Type 1 means identifying a subject prone to, or having, Fredrickson dyslipidemia Type I (a, b, oc), FCS, LPLD. Identification of subjects with Fredrickson Type I, FCS, LPL<d>, can be accomplished by examination of the subject's medical history in conjunction with any screening technique known in the art, e.g., genetic screening or screening for LPL inhibitors. For example, a patient with a documented medical history of fasting TG above 750 mg/dL is then screened for mutations in the LPL gene or genes that affect LPL, such as ApoC2, ApoA5, GPIHBP1, or LMF1.

"Identifying" or "diagnosing" an animal with metabolic or cardiovascular disease means identifying a subject prone to, or having, a metabolic disease, a cardiovascular disease, or a metabolic syndrome; or, identify a subject who has any symptoms of a metabolic disease, cardiovascular disease or metabolic syndrome, including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, weight body fat content above normal and/or body fat content above normal or any combination thereof. Such identification can be achieved by any method, including, but not limited to, standard clinical tests or evaluations, such as measurement of serum or circulating (plasma) cholesterol, editing of serum or circulating (plasma) blood glucose, measurement of triglycerides. in serum or circulating (plasma), measurement of blood pressure, measurement of body fat content, measurement of body weight, and the like.

"Improved cardiovascular outcome" means a reduction in the occurrence of, or risk of, adverse cardiovascular events. Examples of adverse cardiovascular events include, but are not limited to, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial arrhythmia.

"Immediately adjacent" means that there are no intermediate elements between immediately adjacent elements, for example, between regions, segments, nucleotides and/or nucleosides.

"Increase HDL" or "raise HDL" means increasing the level of HDL in an animal after administration of at least one compound of the invention, compared to the level of HDL in an animal that has not been administered any compound.

"Individual" or "subject" or "animal" means a human or non-human animal selected for treatment or therapy.

"Induce", "inhibit", "enhance", "raise", "increase", "decrease", "reduce" or the like denote quantitative differences between two states. For example, "an amount effective to inhibit the activity or expression of ApoCIII" means that the level of activity or expression of ApoCIII in a treated sample will differ from the level of activity or expression of ApoCIII in an untreated sample. Such terms apply to, for example, levels of expression, and levels of activity.

"Inhibit expression or activity" refers to a reduction or blockage of the expression or activity of an RNA or protein and does not necessarily indicate a complete elimination of expression or activity.

"Insulin resistance" is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response of fat, muscle, and liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in the muscle reduces glucose uptake, while insulin resistance in the liver reduces glucose storage, both effects serving to raise blood glucose. High plasma levels of insulin and glucose due to insulin resistance often lead to metabolic syndrome and type 2 diabetes.

"Insulin sensitivity" is a measure of how effectively an individual processes glucose. An individual who has high insulin sensitivity processes glucose efficiently, while an individual with low insulin sensitivity does not process glucose effectively.

"Internucleoside bonds" refers to the chemical bond between nucleosides.

"Intravenous administration" means administration into a vein.

"Linked nucleosides" means adjacent nucleosides that are linked together.

"Lipid lowering" means a reduction in one or more lipids in a subject. "Lipid elevation" means an increase in a lipid (e.g., HDL) in a subject. Lipid lowering or lipid elevation may occur with one or more doses over time.

"Lipid-lowering therapy" or "lipid-lowering agent" means a therapeutic regimen provided to a subject to lower one or more lipids in a subject. In certain embodiments, lipid-lowering therapy is provided to reduce one or more of CETP, ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles , and Lp(a) in a subject. Examples of lipid-lowering therapy include statins, fibrates, MTP inhibitors.

"Lipoprotein", such as VLDL, LDL, and HDL, refers to a group of proteins found in serum, plasma, and lymph that are important for lipid transport. The chemical composition of each lipoprotein differs in that HDL has a higher protein to lipid ratio, while VLDL has a lower protein to lipid ratio.

"Lipoprotein Lipase" or "LPL" refers to an enzyme that hydrolyzes TG found in lipoproteins, such as CM or VLDL, into free fatty acids and monoacylglycerols. ÑA LPL requires apo C-II as a cofactor to function in the hydrolyzation of TG. LPL is produced mainly in skeletal muscle, adipose tissue, and cardiac muscle. Hydrolysis and removal of TG from CM and VLDL normally protects against excessive postprandial increases in CM and TG mass.

"Lipoprotein lipase deficient", "lipoprotein lipase deficiency", "LPL deficiency" or "LPLD" is also known as "Fredrickson dyslipidemia Type I", "chylomicronemia", "Familial Chylomicronemia Syndrome" or "FCS". Although subjects with LPLD generally lack the LPL or LPL activity necessary for the efficient breakdown of fatty acids such as TG, these subjects may still have minimal LPL activity or express a minimal level of LPL. In some cases, a subject with LPLD may express LPL or have LPL activity of up to approximately, or no more than, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12 %, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of activity. In other cases, the subject with LPLD has no LPL or measurable LPL activity. One embodiment of LPLD encompasses subjects with "hyperlipoproteinemia type Ia" (also known as "Fredrickson Type Ia") and refers to the inability of subjects to produce sufficient functional lipoprotein lipase enzymes necessary for the efficient breakdown of fatty acids such as TG. The inability to break down TG leads to hypertriglyceridemia in the subject and, often more than 12 hours after meals, hyperTG and chylomicronemia are still present and visible as lipemia. Type A is commonly caused by one or more mutations in the LPL gene. As disclosed herein, LPLD also encompasses subjects who have dysfunctional lipoprotein lipase, such as subjects with "hyperlipoproteinemia Type lb" (also known as "Fredrickson Type lb") and "hyperlipoproteinemia type Ic" (also known as Fredrickson type IC"). Type lb is caused by lack of the lipoprotein lipase activator apoprotein C-II. Type Ic is due to a circulating lipoprotein lipase inhibitor. As with Type 1a, subjects with Type 1b/1c suffer from an inability to break down TG leading to hypertriglyceridemia and hyperTG and chylomicronemia are still present and visible as lipemia often more than 12 hours after meals. In certain embodiments, LPLD is associated with at least one mutation in the LPL gene such as P207L, G188L or D9N or other mutations that affect LPL (BruBrunzell, In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle 1993-2013 [updated December 15, 2011].

"Low-density lipoprotein-cholesterol (LDL-C)" means cholesterol transported in low-density lipoprotein particles. The concentration of LDL-C in serum (or plasma) is typically quantified in mg/dl or nmol/l. "Serum LDL-C" and "plasma LDL-C" mean serum and plasma LDL-C, respectively.

"Major risk factors" refers to factors that contribute to a high risk of a particular disease or condition. In certain embodiments, major risk factors for coronary heart disease include, without limitation, smoking, hypertension, low HDL-C, family history of coronary heart disease, age, and other factors disclosed herein.

"Metabolic disorder" or "metabolic disease" refers to a condition characterized by an alteration or disturbance in metabolic function. "Metabolic" and "metabolism" are terms well known in the art and generally include the full range of biochemical processes that take place within a living organism. Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome, and dyslipidemia due to type 2 diabetes.

"Metabolic syndrome" means a condition characterized by a cluster of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, the metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference greater than 102 cm in men or greater than 88 cm in women; serum triglycerides of at least 150 mg/dl; HDL-C less than 40 mg/dl in men or less than 50 mg/dl in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 110 mg/dl. These determinants can be easily measured in clinical practice (JAMA, 2001, 285: 2486-2497).

"Matching" or "non-complementary nucleobase" refers to the case in which a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second target nucleic acid.

"Mixed dyslipidemia" means a condition characterized by high cholesterol and high triglycerides. "Modified internucleoside bond" refers to a substitution or any change to a naturally occurring internucleoside bond. For example, a phosphorothioate bond is a modified internucleoside bond.

"Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. For example, 5-methylcytosine is a modified nucleobase. An "unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

"Modified nucleoside" means a nucleoside that has at least a modified sugar and/or modified nucleobase moiety.

"Modified nucleotide" means a nucleotide that has at least one modified sugar moiety, one modified internucleoside bond and/or modified nucleobase.

"Modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleotide.

"Modified sugar" refers to a substitution or change of a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

"Motif" means the pattern of chemically distinct regions in an antisense compound.

"Naturally occurring internucleoside bond" means a 3' to 5' phosphodiester bond.

"Natural sugar moiety" means a sugar found in DNA (2'-H) or RNA (2'-OH).

"Nicotinic acid" or "niacin" has been reported to decrease the influx of fatty acids to the liver and the secretion of VLDL by the liver. This effect appears to be mediated in part by effects on hormone-sensitive lipase in adipose tissue. Nicotinic acid has key sites of action in both the liver and adipose tissue. In the liver, nicotinic acid is reported to inhibit diacylglycerol acyltransferase-2 (DGAT-2) resulting in decreased secretion of VLDL particles from the liver, which is also reflected in reductions of both IDL and LDL particles. Furthermore, nicotinic acid increases HDL-C and apo A1 primarily by stimulating apo A1 production in the liver and has also been shown to reduce VLDL-ApoCIII concentrations in patients with hyperlipidemia Wahlberg et al. Acta Med Scand 1988; 224:319-327). The effects of nicotinic acid on lipolysis and fatty acid mobilization in adipocytes are well established. However, nicotinic acid has not been effective in treating LPLD patients with hypertriglyceridemia.

"Nucleic acid" refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids (ssDNA), double-stranded nucleic acids (dsDNA), small interfering ribonucleic acids (siRNA), and microRNA (miRNA). A nucleic acid may also comprise a combination of these elements in a single molecule.

"Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

"Nucleobase complementarity" refers to a nucleobase that is capable of forming base pairs with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, the complementary nucleobase refers to a nucleobase of an antisense compound that is capable of forming base pairs with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the oligonucleotide and the target nucleic acid are considered complementary in that nucleobase pair.

"Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, bond or nucleobase modifications.

"Nucleoside" means a nucleobase linked to a sugar.

"Nucleoside mimetic" includes those structures used to replace the sugar or sugar and base, and not necessarily bonding at one or more positions of an oligomeric compound; for example, nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics as non-furanose sugar units.

"Nucleotide" means a nucleoside that has a phosphate group covalently linked to the sugar portion of the nucleoside.

"Nucleotide mimetic" includes those structures used to replace the nucleoside and linker at one or more positions of an oligomeric compound such as, for example, peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=O) -O- or other non-phosphodiester bond).

"Oligomeric compound" or "oligomer" means a polymer of linked monomeric subunits that is capable of hybridizing with a region of a nucleic acid molecule. In certain embodiments, the oligomeric compounds are oligonucleosides. In certain embodiments, the oligomeric compounds are oligonucleotides. In certain embodiments, the oligomeric compounds are antisense compounds. In certain embodiments, the oligomeric compounds are antisense oligonucleotides. In certain embodiments, the oligomeric compounds are chimeric oligonucleotides.

"Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independently of each other.

"Parenteral administration" means administration via injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration or intracranial administration, for example, intrathecal or intracerebroventricular administration. Administration can be continuous, chronic, short or intermittent.

"Peptide" means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.

"Pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeting ApoCIII is a pharmaceutical agent.

"Pharmaceutical composition" or "composition" means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may comprise one or more active agents and a pharmaceutical carrier, such as a sterile aqueous solution.

"Pharmaceutically acceptable carrier" means a medium or diluent that does not interfere with the structure of the compound. Some such carriers allow pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral ingestion by a subject. Some such carriers allow pharmaceutical compositions to be formulated for injection, infusion or topical administration. For example, a pharmaceutically acceptable carrier may be a sterile aqueous solution.

"Pharmaceutically acceptable derivative" or "salts" encompasses derivatives of the compounds described herein as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labeled variants, pharmaceutically acceptable salts and other derivatives known in the art.

"Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, that is, salts that retain the desired biological activity of the starting compound and do not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" includes a salt prepared from non-toxic pharmaceutically acceptable acids or bases, including inorganic or organic acids and bases. The "pharmaceutically acceptable salts" of the compounds described herein can be prepared by methods well known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and well accepted for therapeutic administration to humans. Accordingly, in one embodiment, the compounds described herein are in the form of a sodium salt.

"Phosphorothioate bond" means a bond between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate bond is a modified internucleoside bond.

"Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

"Prevent" refers to delaying or anticipating the onset or development of a disease, disorder or condition for a period of time from minutes to indefinitely. Prevention also means reducing the risk of developing a disease, disorder or condition.

"Prodrug" means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., a drug) within the body or cells thereof through the action of endogenous enzymes or other chemical products or conditions.

"Elevate" means to increase in quantity. For example, raising plasma HDL levels means increasing the amount of HDL in the plasma.

"TG to HDL ratio" means TG levels in relation to HDL levels. The occurrence of high TG and/or low HDL has been linked to cardiovascular disease incidence, outcomes, and mortality. "Improving the TG to HDL ratio" means decreasing TG levels and/or increasing HDL levels.

"Reduce" means to decrease to a smaller measurement, size, quantity or number. For example, reducing plasma triglyceride levels means decreasing the amount of triglycerides in the plasma.

"Region" or "target region" is defined as a part of the target nucleic acid that has at least one identifiable structure, function or characteristic. For example, a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid region. Structurally defined regions for ApoCIII can be obtained by the accession number of sequence databases such as NCBI. In certain embodiments, a target region may span the sequence from a target site 5' of a target segment within the target region to a target site 3' of another target segment within the target region.

"Ribonucleotide" means a nucleotide that has a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides can be modified with any of a variety of substituents.

"Second agent" or "second therapeutic agent" means an agent that can be used in combination with a "first agent." A second therapeutic agent may include, but is not limited to, an siRNA or antisense oligonucleotide including antisense oligonucleotides targeting ApoCIII. A second agent may also include anti-ApoCIII antibodies, ApoCIII peptide inhibitors, cholesterol-lowering agents, lipid-lowering agents, glucose-lowering agents and anti-inflammatory agents.

"Segments" are defined as smaller sub-portions of regions within a nucleic acid. For example, a "target segment" means the nucleotide sequence of a target nucleic acid to which one or more antisense compounds are directed. "Target site 5'"' refers to the 5' most nucleotide of a target segment. "Target site '"' refers to the 3' most nucleotide of a target segment.

The "shortened" or "truncated" versions of antisense oligonucleotides or target nucleic acids taught herein have one, two or more nucleosides deleted.

"Side effects" means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, kidney function abnormalities, liver toxicity, kidney toxicity, central nervous system abnormalities, myopathies, and malaise. For example, elevated serum aminotransferase levels may indicate liver toxicity or abnormality of liver function. For example, increased bilirubin may indicate liver toxicity or abnormality of liver function.

"Single-stranded oligonucleotide" means an oligonucleotide that is not hybridized to a complementary strand.

"Specifically hybridizable" refers to an antisense compound that has a sufficient degree of complementarity with a target nucleic acid to induce a desired effect, while showing minimal or no effects on non-target nucleic acids under conditions in which the specific binding, that is, under physiological conditions in the case of in vivo assays and therapeutic treatments.

"Statin" means an agent that inhibits the activity of HMG-CoA reductase. Statins reduce cholesterol synthesis in the liver by competitively inhibiting HMG-CoA reductase activity. Reduction in intracellular cholesterol concentration induces LDL receptor expression on the cell surface of hepatocytes, resulting in increased removal of LDL-C from the blood and reduced concentration of circulating LDL-C and other lipoproteins. containing apo-B including TG-rich particles. Independently of their effects on LDL-C and the LDL receptor, statins reduce the plasma concentration and cellular mRNA levels of ApoC-III (Ooi et al. Clinical Sci, 2008, 114:611-624). As statins have significant effects on mortality as well as most parameters of cardiovascular disease outcomes, these drugs are the first choice to reduce both the risk of total cardiovascular disease and moderately elevated TG levels. The most potent statins (atorvastatin, rosuvastatin, and pitavastatin) demonstrate a robust decrease in TG levels, especially at high doses and in patients with high TG. However, statins have been ineffective in treating LPLD patients with hypertriglyceridemia.

"Subcutaneous administration" means administration just under the skin.

"Subject" means a human or non-human animal selected for treatment or therapy.

"Symptom of cardiovascular disease or disorder" means a phenomenon that arises from and accompanies the cardiovascular disease or disorder and serves as an indication thereof. For example, angina pectoris; chest pain; difficulty breathing; palpitations; weakness; dizziness; nausea; perspiration; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; cyanosis; fatigue; Fainting; numbness of the face; numbness of the extremities; claudication or muscle cramps; swelling of the abdomen; or fever are symptoms of cardiovascular disease or disorder.

"Targeting" or "targeting" means the process of designing and selecting an antisense compound that will specifically hybridize with a target nucleic acid and induce a desired effect.

"Target nucleic acid", "target RNA" and "target RNA transcript" all refer to a nucleic acid capable of being targeted by antisense compounds.

"Therapeutic lifestyle change" means dietary and lifestyle changes aimed at decreasing fat mass/adipose tissue and/or cholesterol. Such a change may reduce the risk of developing heart disease, and may include recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrates, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity.

"Treat" refers to administering a compound of the invention to effect an alteration or improvement of a disease, disorder or condition.

"Triglyceride" or "TG" means a neutral lipid or fat consisting of glycerol combined with three fatty acid molecules.

"Type 2 diabetes" (also known as "type 2 diabetes mellitus", "diabetes mellitus, type 2", "non-insulin-dependent diabetes (NIDDM)", "obesity-related diabetes", or "adult-onset diabetes" ) is a metabolic disorder primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.

"Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties and internucleoside bonds. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e., p-D-ribonucleoside) or a DNA nucleotide (i.e., p-D-deoxyribonucleoside).

"Wing segment" means one or a plurality of nucleosides modified to impart to an oligonucleotide properties such as improved inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by nucleases in vivo.

Certain realizations

Disclosed herein is a method of reducing ApoCIII levels in an animal with Fredrickson Dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal. In certain embodiments, ApoCIII levels are reduced in the liver, adipose tissue, heart, skeletal muscle, or small intestine.

Certain embodiments provide a compound comprising a specific inhibitor of ApoCIII for use in the invention for use in the treatment, prevention, delay or amelioration of Fredrickson dyslipidemia Type I, FCS, LPLD, in an animal. In certain cases, a cardiovascular and/or metabolic disease or disorder, or symptom or risk thereof, related to Fredrickson dyslipidemia Type I, FCS, LPLD is improved.

Certain embodiments provide a compound comprising a specific inhibitor of ApoCIII for use in the invention for use in the treatment, prevention, delay or amelioration of pancreatitis in an animal with Fredrickson's Dyslipedia Type I, FCS, LPLD. In certain embodiments, pancreatitis or a symptom or risk thereof is improved.

Disclosed herein is a method of reducing TG levels in an animal with Fredrickson's Dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal.

In certain embodiments, the animal has a TG level of at least >1200mg/dl, >1100mg/dl, >1000mg/dl, >900mg/dl, >880mg/dl, >850mg/dl, >800mg/dl, >750mg/dl, >700mg/dl, >650mg/dl, >600mg/dl, >550mg/dl, >500mg/dl, >450mg/dl, >440mg/dl, >400mg/dl, >350mg/dl, >300mg/dl, >250mg/dl, >200mg/dl, >150mg/dl. In certain embodiments, the animal has a history of TG level >880 mg/dL, fasting TG level >750 mg/dL, and/or post-diet TG level >440 mg/dL.

In certain cases, the compound decreases TG (postprandial or fasting) by at least 90%, at least 80%, at least 70%, at least 60%, at least 50% , at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5% or at least 1% % of the reference TG level. In certain embodiments, the TG level (postprandial or fasting) is <1900mg/dl, <1800mg/dl, <1700mg/dl, <1600mg/dl, <1500mg/dl, <1400mg/dl, <1300mg/dl, <1200mg/dl, <1100mg/dl, <1000mg/dl, <900mg/dl, <800mg/dl, <750mg/dl, <700mg/dl, <650mg/dl, <600mg/dl, <550mg/dl, <500mg/dl, <450mg/dl, <400mg/dl, <350mg/dl, <300mg/dl, <250mg/dl, <200mg/dl, <150mg/dl or <100mg/dl.

Disclosed herein is a method of increasing HDL levels and/or improving the TG to HDL ratio in an animal with Fredrickson dyslipidemia type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal. In certain embodiments, the compound increases HDL (postprandial or fasting) by at least 90%, at least 80%, at least 70%, at least 60%, at least 50% , at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5% or at least 1% % from the reference HDL level.

In certain embodiments, the compound decreases ApoCIII by about 81%, decreases TG by about 69%, decreases VLDL ApoClII by about 80%, increases HDL by about 78%, decreases non-HDL-C by approximately 58% and/or decreases ApoB by approximately 13%.

Disclosed herein is a method of preventing, delaying or ameliorating a disease, disorder, cardiovascular and/or metabolic condition or symptom thereof, in an animal with Fredrickson's Dyslipidemia Type I, FCS, LPLD, comprising therapeutically administering an amount effective of a compound comprising a specific inhibitor of ApoCIII to the animal. In certain cases, the compound prevents, delays or improves the disease, disorder, cardiovascular and/or metabolic condition or symptom thereof in the animal with Fredrickson dyslipidemia Type I, FCS, LPLD, decreasing TG levels, increasing levels of HDL in the animal and/or improving the ratio of TG to HDL.

Certain embodiments provide a compound comprising a specific ApoCIII inhibitor for use in the invention to prevent, delay or ameliorate pancreatitis, or symptom thereof, in an animal with Fredrickson dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising an ApoCIII inhibitor specific to the animal. In certain embodiments, the compound prevents, delays or improves pancreatitis, or symptom thereof, in the animal with Fredrickson's dyslipidemia Type I, FCS, LPLD, by decreasing TG levels, increasing HDL levels in the animal and/ or improving the ratio of TG to HDL.

Disclosed herein is a method of preventing, delaying or ameliorating pancreatitis, or symptoms thereof, in an animal with Fredrickson's dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising an inhibitor. ApoCIII specific to the animal. In certain embodiments, the compound prevents, delays or improves pancreatitis, or symptom thereof, in the animal with Fredrickson's dyslipidemia Type I, FCS, LPLD, by decreasing TG levels, increasing HDL levels in the animal and/ or improving the ratio of TG to HDL.

Disclosed herein is a method of preventing, treating, ameliorating, delaying the onset, or reducing the risk of a cardiovascular disease, disorder or condition in an animal with Fredrickson's Dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically administered amount effective of a compound comprising a specific inhibitor of ApoCIII to the animal. In certain embodiments, the compound prevents, treats, improves, delays the onset or reduces the risk of the cardiovascular disease, disorder or condition in the animal with Fredrickson dyslipidemia Type I, FCS, LPLD, by decreasing TG levels, increasing TG levels. of HDL and/or improving the ratio of TG to HDL.

Disclosed herein is a method for decreasing levels of CETP, VLDL, VLDL ApoCIII, cholesterol, chylomicrons and/or ApoB in an animal with Fredrickson's dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound that It comprises an animal-specific inhibitor of ApoCIII. In certain embodiments, the ApoB is ApoB-48 or ApoB-100. In certain embodiments, the amount of ApoB-48 reflects the amount of chylomicrons in the animal. In certain embodiments, the cholesterol is total cholesterol or non-HDL cholesterol.

Disclosed herein is a method of increasing ApoA1, PON1, fat clearance, chylomicrontriglyceride (CM-TG) clearance and/or HDL in an animal with Fredrickson Dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal. Disclosed herein is a method for improving the ratio of TG to HDL in an animal with Fredrickson's Dyslipidemia Type I, FCS, LPLD comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the animal.

Disclosed herein is a method of treating adult patients with Fredrickson's dyslipidemia type I, FCS, LPLD suffering from severe or multiple attacks of pancreatitis comprising administering a therapeutically effective amount of a compound comprising a specific ApoCIII inhibitor to the patient. In certain embodiments, the patient suffers from pancreatitis despite dietary fat restrictions.

Disclosed herein is a method for identifying a subject suffering from Fredrickson Dyslipidemia Type I, FCS, LPLD, which comprises genetically screening the subject.

Disclosed herein is a method for identifying a subject at risk for Fredrickson's Dyslipidemia Type I, FCS, LPLD, which comprises genetically screening the subject. In certain embodiments, genetic screening is performed by sequence analysis of the RA gene or transcript encoding LPL or ApoC-II. In certain embodiments, the subject is genetically screened for at least one mutation in the LPL gene such as P207L, G188L, D9N or other mutations affecting LPL (Brunzell, In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, editors. GeneReviews™ [Internet].

Disclosed herein is a method for identifying a subject suffering from Fredrickson Dyslipidemia Type I, FCS, LPLD, which comprises screening the subject for the presence of LPL-inhibiting antibodies. Disclosed herein is a method for identifying a subject at risk for Fredrickson's dyslipidemia Type I, FCS, LPLD, which comprises screening the subject for the presence of LPL-inhibiting antibodies.

In certain embodiments, the level of LPL expression in a subject with LPLD is undetectable. In certain embodiments, the level of LPL in a subject with LPLD is detectable. In certain embodiments, the LPL level in the subject with LPLD is at most 25%, at most 24%, at most 23%, at most 22%, at most 21%, at most 20%, at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3 % at most 2% or at most 1% of the LPL level of a person who does not have LPLD.

In certain embodiments, the level of LPL activity in a subject with LPLD is undetectable. In certain embodiments, the level of LPL activity in a subject with LPLD is detectable. In certain embodiments, the level of LPL activity in the LPLD subject is at most 25%, at most 24%, at most 23%, at most 22%, at most 21%, at most 20 %, at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1% of the LPL activity level of a subject without LPLD. In certain embodiments, the ApoCIII nucleic acid is any of the sequences set forth in GENBANK Accession No. NM 000040.1 (incorporated herein as Eq ID NO: 1), GENBANK Accession No. NT 033899. 8 truncated from the nucleotides 20262640 to 20266603 (incorporated herein as SEQ ID NO: 2), and GenBank Accession No. NT 035088.1 truncated from nucleotides 6238608 to 6242565 (incorporated herein as SEQ ID NO: 4).

In certain embodiments, the specific ApoCIII inhibitor is an RNA interfering compound capable of inhibiting ApoCIII expression. In certain embodiments, the compound comprises at least one modified internucleoside bond. In certain embodiments, the internucleoside bond is a phosphorothioate internucleoside bond. In certain embodiments, each internucleoside bond is a phosphorothioate internucleoside bond.

In certain embodiments, the compound comprises at least one nucleoside comprising a modified sugar. In certain embodiments, the at least one modified sugar is a bicyclic sugar. In certain embodiments, the at least one modified sugar comprises a 2'-O-methoxyethyl.

In certain embodiments, the compound comprises at least one nucleoside comprising a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the compound comprises a modified oligonucleotide comprising: (i) a gap segment consisting of linked deoxynucleosides; (ii) a 5' wing segment consisting of linked nucleosides; (iii) a 3' wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

Symptoms of cardiovascular disease include, but are not limited to, angina; chest pain; difficulty breathing; palpitations; weakness; dizziness; nausea; perspiration; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; cyanosis; fatigue; Fainting; numbness of the face; numbness of the extremities; claudication or muscle cramps; swelling of the abdomen; or fever.

In certain embodiments, the animal is human.

In certain cases, cardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia or hypercholesterolemia. In certain embodiments, the dyslipidemia is hypertriglyceridemia or chylomicronemia (e.g., FCS). In certain embodiments, the metabolic disease is diabetes, obesity or metabolic syndrome.

In certain embodiments, the animal with Fredrickson's dyslipidemia Type I, FCS, LPLD, is at risk for pancreatitis. In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine prevents pancreatitis. In certain embodiments, lowering TG levels, raising HDL levels, and/or improving the ratio of TG to HDL prevents pancreatitis.

In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine of an animal with Fredrickson Dyslipidemia Type I, FCS, LPLD, improves postprandial TG clearance. In certain embodiments, raising HDL levels and/or improving the TG to HDL ratio improves postprandial TG clearance in an animal with Fredrickson Dyslipidemia Type I, FCS, LPLD. In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine reduces postprandial triglyceride in an animal with Fredrickson dyslipidemia Type I, FCS, LPLD. In certain embodiments, raising HDL levels and/or improving the ratio of TG to HDL decreases postprandial TG.

In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine of an animal with Fredrickson Dyslipidemia Type I, FCS, LPLD, improves the ratio of HDL to TG.

In certain embodiments, the compound is administered parenterally. In additional embodiments, the parenteral administration is subcutaneous.

In certain embodiments, the compound is coadministered with a second agent or therapy. In certain embodiments, the second agent is an ApoCIII lowering agent, Apo C-II lowering agent, DGAT1 lowering agent, LPL raising agent, cholesterol lowering agent, non-HDL lipid lowering agent, LDL lowering agent, agent TG lowering, cholesterol lowering agent, HDL raising agent, fish oil, niacin (nicotinic acid), fibrate, statins, DCCR (diazoxide salt), glucose lowering agent or antidiabetic agents. In certain embodiments, the second therapy is dietary fat restriction.

In certain embodiments, the ApoCIII reducing agents include an ApoCIII antisense oligonucleotide different from the first agent, fibrate, or an Apo B antisense oligonucleotide.

In certain embodiments, the DGAT1 reducing agent is LCQ908.

In certain embodiments, LPL-raising agents include gene therapy agents that raise the level of LPL (e.g., GlyberaR, normal copies of ApoC-II, GPIHBP1, APOA5, LMF1 or other genes that, when mutated, can lead to LPL dysfunctional).

In certain embodiments, glucose-lowering and/or antidiabetic agents include, but are not limited to, PPA agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or an insulin analog, a secretagogue. insulin, an SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, metformin, sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione, alpha-glucosidase inhibitor and the like. The sulfonylurea may be acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide or a gliclazide. Meglitinide can be nateglinide or repaglinide. The thiazolidinedione can be pioglitazone or rosiglitazone. Alpha-glucosidase can be acarbose or miglitol.

In certain embodiments, cholesterol or lipid lowering agents include, but are not limited to, statins, bile acid sequestrants, nicotinic acid, and fibrates. Statins may be atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin and the like. Bile acid sequestrants may be colesevelam, cholestyramine, colestipol and the like. Fibrates can be gemfibrozil, fenofibrate, clofibrate and the like. Therapeutic lifestyle change may be dietary fat restriction.

In certain embodiments, agents that increase HDL include drugs that inhibit cholesteryl ester transfer protein (CETP) (such as Torcetrapib), peroxisome proliferator-activated receptor agonists, Apo-A1, Pioglitazone and the like.

In certain embodiments, the compound and the second agent are administered concomitantly or sequentially.

In certain embodiments, the compound is a form of salt. In further embodiments, the compound further comprises a pharmaceutically acceptable carrier or diluent.

Antisense compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides and siRNA. An oligomeric compound may be "antisense" to a target nucleic acid, meaning that it is capable of undergoing hybridization with a target nucleic acid through hydrogen bonding.

The antisense compounds provided herein refer to oligomeric compounds capable of hybridizing with a target nucleic acid via hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as antisense oligonucleotides, siRNA, shRNA, and miRNA.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is directed. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is directed.

In certain embodiments, an antisense compound targeting an ApoCIII nucleic acid is 12 to 30 nucleotides in length. In other words, antisense compounds are 12 to 30 nucleobases linked. In other embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8 to 80, 10 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22 or 20 linked nucleobases.

When a single additional nucleoside is present in an elongated oligonucleotide, the additional nucleoside may be located in the central portion, the 5' or 3' end of the oligonucleotide. When two or more additional nucleosides are present, the added nucleosides may be adjacent to each other, for example, in an oligonucleotide that has two nucleosides added to the central portion, to the 5' end (5' addition) or alternatively to the 3' end ( 3' addition) of the oligonucleotide. Alternatively, the added nucleosides can be dispersed throughout the antisense compound, for example, in an oligonucleotide having a nucleoside added to the 5' end and a subunit added to the 3' end.

It is possible to increase or decrease the length of an antisense compound and/or introduce mismatched bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an injection model. of oocytes. Antisense oligonucleotides of 25 nucleobases in length with 8 or 11 bases mismatches near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, although to a lesser extent than antisense oligonucleotides that did not contain mismatches. Similarly, target-specific cleavage was achieved using 13-nucleotide antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide that has 100% complementarity with the bcl-2 mRNA and that has 3 mismatches with the bcl-2 mRNA xL to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent antitumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested a series of 14-nucleobase antisense oligonucleotides in tandem, and 28- and 42-nucleobase antisense oligonucleotides composed of the sequence of two or three of the antisense oligonucleotides. in tandem, respectively, for their ability to stop translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14-nucleobase antisense oligonucleotides alone was able to inhibit translation, although to a more modest level than the 28- or 42-nucleobase antisense oligonucleotides.

Motifs of Antisense Compounds

In certain embodiments, antisense compounds targeting an APoCIII nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer the antisense compounds with properties such as improved inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by nucleases in vivo.

Chimeric antisense compounds typically contain at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid and/or increased inhibitory activity. A second region of a chimeric antisense compound can optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense compounds that have a gapmer motif are considered chimeric antisense compounds. In a gapmer, an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as a substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, regions of a gapmer are differentiated by the types of sugar moieties that comprise each distinct region. The types of sugar moieties that are used to differentiate regions of a gapmer may in some embodiments include p-D-ribonucleosides, p-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE, and 2'-MOE, and '-O-CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4'-(CH2)n-O-2' bridge, where n=1 or n=2). Preferably, each distinct region comprises uniform sugar fractions. The wing-gap-wing motif is frequently described as "X-Y-Z", where "X" represents the length of the 5' wing region, "Y" represents the length of the gap region, and "Z" represents the length of the 3' wing region. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the gap segment is positioned immediately adjacent to each of the 5' wing segment and the 3' wing segment. Therefore, there are no intermediate nucleotides between the 5' wing segment and the gap segment, or the gap segment and the 3' wing segment. Any of the antisense compounds described herein may have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. * , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. Therefore, gapmers include, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5 , 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6 8-6, 5-8-5, 1- 8-1, 2-6-2, 2-13-2, 1-8-2, 2-8-3, 3-10-2, 1-18-2 or 2-18-2.

In certain embodiments, the antisense compound is a "wingmer" motif, which has a gapmer or gap-wing configuration, i.e., an X-Y or Y-Z configuration as described above for the gapmer configuration. Therefore, wingmer configurations include, but are not limited to, for example 5-10, 8-4, 4 12, 12-4, 3-14, 16-2, 18-1, 10-3, 2- 10, 1-10, 8-2, 2-13, or 5-13.

In certain embodiments, antisense compounds targeting an ApoCIII nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, an antisense compound targeting an ApoCIII nucleic acid has an extended gap motif.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences encoding ApoCIII include, without limitation, the following: GENBANK Accession No. NM_000040.1 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_033899.8 truncated nucleotides 20262640 to 20266603 (incorporated herein as SEQ ID NO: 2) and GenBank accession No. NT_035088.1 truncated from nucleotides 6238608 to 6242565 (incorporated herein as SEQ ID NO: 4).

It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside bond, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may independently comprise one or more modifications to a sugar moiety, an internucleoside bond, or a nucleobase. Antisense compounds described by Isis Number (Isis No.) indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, a translation termination region or another defined nucleic acid region. Structurally defined regions for ApoCIII can be obtained by the accession number of sequence databases such as NCBI. In certain embodiments, a target region may span the sequence from a target site 5' of a target segment within the target region to a target site 3' of another target segment within the target region.

In certain embodiments, a "target segment" is a smaller, subportion of a target region within a nucleic acid. For example, a target segment may be the nucleotide sequence of a target nucleic acid to which one or more antisense compounds are directed. The "5' target site" refers to the 5' most nucleotide of a target segment. The "3' target site" refers to the 3' most nucleotide of a target segment.

A target region may contain one or more target segments. Multiple target segments may overlap within a target region. Alternatively, they may not overlap. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is approximately, is not more than, is not more than approximately, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides in the target nucleic acid, or is an interval defined by any two of the above values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides in the target nucleic acid. In certain embodiments, the target segments are contiguous. Target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or the 3' target sites listed herein are contemplated.

Targeting includes determining at least one target segment to which an antisense compound hybridizes such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in the levels of the mRNA target nucleic acid. In certain embodiments, the desired effect is reduction in levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

Suitable target segments can be found within a 5' UTR, a coding region, a 3' UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or the stop codon.

Determination of suitable target segments may include a comparison of the sequence of a target nucleic acid with other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison can avoid the selection of antisense compound sequences that may hybridize in a non-specific manner with sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

There may be variation in the activity (for example, as defined by the percentage reduction in target nucleic acid levels) of antisense compounds within an active target region. In certain embodiments, reductions in ApoCIII mRNA levels are indicative of inhibition of ApoCIII expression. Reductions in the levels of an ApoCIII protein are also indicative of inhibition of target mRNA expression. Furthermore, phenotypic changes may be indicative of inhibition of ApoCIII expression. For example, an increase in the HDL level, a decrease in the LDL level or a decrease in the TG level are among the phenotypic changes that can be assayed for the inhibition of ApoCIII expression. Other phenotypic indications may also be evaluated, for example, symptoms associated with cardiovascular or metabolic disease; for example, angina pectoris; chest pain; difficulty breathing; palpitations; weakness; dizziness; nausea; perspiration; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; cyanosis; fatigue; Fainting; numbness of the face; numbness of the extremities; claudication or muscle cramps; swelling of the abdomen; or fever. Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and an ApoCIII nucleic acid. The most common mechanism of hybridization involves hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or inverted Hoogsteen hydrogen bonds) between complementary nucleobases of nucleic acid molecules.

Hybridization can take place under different conditions. The stringent conditions are sequence dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, CSHL Press). In certain embodiments, the antisense compounds provided herein are specifically hybridizable to an ApoCIII nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond to the corresponding nucleobases of the target nucleic acid such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as an ApoCIII nucleic acid).

An antisense compound may hybridize to one or more segments of an ApoCIII nucleic acid such that intermediate or adjacent segments are not involved in the hybridization event (e.g., a turn, mismatch, or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an ApoCIII nucleic acid, a target region, a target segment , or a specified portion thereof. The percentage complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore hybridize specifically, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be clustered or interspersed with complementary nucleobases and do not need to be contiguous with each other or with complementary nucleobases. As such, an antisense compound that is 18 nucleobases in length that has 4 (four) non-complementary nucleobases that are flanked by two regions of complete complementarity with the target nucleic acid would have a total complementarity of 77.8% with the target nucleic acid and would therefore fall within the scope of the present invention. The percent complementarity of an antisense compound to a region of a target nucleic acid can be routinely determined using BLAST (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403410; Zhang and Madden, Genome Res., 1997, 7, 649656). The percentage of sequence homology, identity or complementarity can be determined by, for example, the program Gap (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison W es), using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482489).

In certain embodiments, the antisense compounds provided herein, or specific portions thereof, are completely complementary (i.e., 100% complementary) to a target nucleic acid, or a specific portion thereof. For example, an antisense compound may be completely complementary to an ApoCIII nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means that each nucleobase of an antisense compound is capable of precisely base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20-nucleobase antisense compound is completely complementary to a target sequence that is 400 nucleobases in length, as long as there is a corresponding 20-nucleobase portion of the target nucleic acid that is completely complementary to the antisense compound. Completely complementary may also be used in reference to a specified portion of the first and/or second nucleic acid. For example, a 20-nucleobase portion of a 30-nucleobase antisense compound may be "fully complementary" to a target sequence that is 400 nucleobases in length. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is completely complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion in which each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30-nucleobase antisense compound may or may not be completely complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase(s) may be at the 5' end or at the 3' end of the antisense compound. Alternatively, the non-complementary nucleobase(s) may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous.

In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an ApoCIII nucleic acid, or a specified portion thereof.

In certain embodiments, antisense compounds having, or having up to 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an ApoCIII nucleic acid, or a specified portion thereof.

Antisense compounds provided herein also include those that are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. A "portion" may also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds are complementary to at least an 8-nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10-nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12-nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to a portion of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobases of a target segment, or a range. defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity with a particular nucleotide sequence, SEQ ID NO, or the sequence of a compound represented by a specific Isis number, or a portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and elongated versions of the antisense compounds described herein are contemplated as well as compounds that have non-identical bases relative to the antisense compounds provided herein. Non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. The percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which they are being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identical to one or more of the antisense compounds or SEQ ID NO, or a portion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is typically a heterocyclic base moiety. Nucleotides are nucleosides that also include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent bonding of adjacent nucleosides to each other, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, phosphate groups are commonly referred to as forming internucleoside bonds of the oligonucleotide.

Modifications of antisense compounds include substitutions or changes in internucleoside bonds, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms due to desirable properties such as, for example, improved cellular uptake, improved affinity for the nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be used to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have chemically modified nucleosides.

Modified Internudeosidic Links

The naturally occurring internucleoside bond of RNA and DNA is a 3' to 5' phosphodiester bond. Antisense compounds having one or more modified internucleoside bonds, i.e. of non-natural origin, are often selected over antisense compounds having internucleoside bonds of natural origin due to desirable properties such as, for example, improved cellular uptake, improved affinity for acids. target nucleics and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside bonds include internucleoside bonds that retain a phosphorus atom as well as internucleoside bonds that do not have a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparing phosphorus-containing and non-phosphorus-containing bonds are well known.

In certain embodiments, antisense compounds targeting an ApoCIII nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside bonds are phosphorothioate bonds. In certain embodiments, each internucleoside bond of an antisense compound is a phosphorothioate internucleoside bond.

Modified Sugar Fractions

The antisense compounds of the invention may optionally contain one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may impart improved nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, the nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include, without limitation, the addition of substituent groups (including 5' and 2' substituent groups, bridging of nongeminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ring oxygen atom ribosyl with S, N(R) or C(R-i)(R)<2>(R, R<1>, and R<2>are each independently H, C<1>-C<12>alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published 08/21/08 for other 5' nucleosides, 2'-bis substituted disclosed) or replacement of the oxygen atom of the ribosyl ring by S with additional substitution at the 2' position (see published United States Patent Application US2005-0130923, published June 16, 2005) or alternatively 5' substitution of a BNA (see PCT International Application WO 2007/134181 Published 11/22/07 in which the LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).

Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F,<2>-OCH substituent groups. <3>,<2>-OCH<2>CH<3>, 2'-OCH<2>CH<2>F and 2'-O(CH2)2OCH3. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-C<1>-C<10>alkyl, OCF<3>, OCH<2>F, O(CH<2 >)<2>SCH<3>, O(CH<2>)<2>-O-N(Rm)(Rn), O-CH<2>-C(=O)-N(Rm)(Rn), and O-CH<2>-C(=O)-N(R-,MCH<2>)<2>-N(RmXRn), where each R<1>,Rm and Rn is independently H or C alkyl <1>-C<10>substituted or not substituted.

As used herein, "bicyclic nucleosides" refers to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNA) include, without limitation, nucleosides that comprise a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2' (and analogs thereof see US Patent 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' (and analogs thereof see PCT/US2008/068922 published as WO/2009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' (and analogs thereof see PCT/US2008/064591 published as WO/2008/150729, published December 11, 2008); 4'-CH2-O-N(CH3)-2' (see United States Patent Application US2004-0171570, published September 2, 2004); 4'-C<h>2-N(R)-O-2', where R is H, C<1>-C<12>alkyl, or a protecting group (see US Patent 7,427. 672, issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al, J. Org Chem, 2009, 74, 118-134..); and 4'-CH2-C-(=CH2)-2' (and analogs thereof, see PCT/US2008/066154 published as WO 2008/154401, published December 8, 2008).

Additional bicyclic nucleosides have been reported in the published literature (see, for example, Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; , Curr. Opinion Mol., 2001, 3, 239-243; 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; J. Org., 1998, 63, 10035-10039; 6,268 .490; US2008-0039618; US2004-0171570; ; 61/056,564 ; 61/026,998; 60/989,574; International Applications WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226; and PCT International Applications No. PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591). Each of the above bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and p-D-ribofuranose (see PCT International Application PCT/DK98/00393, published March 25, 1999 as WO 99/ 14226).

As used herein, "monocyclic nucleosides" refers to nucleosides that comprise modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, "4'-2'' bicyclic nucleoside" or "4'-2' bicyclic nucleoside" refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms. of the furanose ring connecting the 2' carbon atom and the 4' carbon atom of the sugar ring.

In certain embodiments, the bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety including, without limitation , bridges comprising 1 or 1 to 4 independently linked groups selected from [C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C (=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)<2>-, -S(=O)x-, and -N(Ra )-; where: x is 0, 1 or 2; n is 1, 2, 3 or 4; each Ra and Rb, is, independently, H, a protecting group, hydroxyl, C<1>-C<12>alkyl, substituted C<1>-C<12>alkyl, C<2>-C<12>alkenyl , substituted C<2>-C<12>alkenyl, C<2>-C<12>alkynyl, substituted C<2>-C<12>alkynyl, C<5>-C<20>aryl, C<aryl 5>-C<20>substituted, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C<5>-C<7>alicyclic radical, C<5>-C<7>substituted alicyclic radical, halogen, OJ< 1>, NJ<1>J<2>, SJ<1>, N<3>, COOJ<1>, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) <2>-J-i) or sulfoxyl (S(=O)-J-i); and

each J<1>and J<2>is, independently, H, C<1>-C<12>alkyl, substituted C<1>-C<12>alkyl, C<2>-C<12>alkenyl, C<2>-C<12>alkenyl substituted, C<2>-C<12>alkynyl, C<2>-C<12>alkynyl substituted, C<5>-C<20>aryl, C<5aryl >-C<20>substituted, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C<1>-C<12> aminoalkyl, C<1>-C< aminoalkyl 12>substituted or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is, -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, -C(RaRb)-N(R) -O- or -C(RaRb)-O-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(C<h>2)2-2', 4'-(CH2)3-2', 4'-CH<2>-O -<2>', 4'-(CH2)2-O-2', 4'-CH2-O-N(R)-2' and 4'-CH2-N(R)-O-2'- where each R is, independently, H, a protecting group or C<1>-C<12>alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-(CH2)-O-2' bridge may be in the a-L configuration or in the p-D configuration. Previously, α-L-methyleneoxy (4'-CH2-O-2') BNA has been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21,6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4' to 2' bridge wherein said bridges include, without limitation, a-L-4'-(CH2)-O-2', p-D-4'-CH2-O- 2', 4'-(CH2)2-O-2', 4'-CH2-O-N(R)-2', 4'-CH2-N(R)-O-2', 4'-CH(CH3 )-O-2', 4'-CH2-S-2', 4'-CH2-N(R)-2', 4'-CH2-CH(CH3)-2', and 4'-(CH2) a-2', where R is H, a protecting group or C<1>-C<12>alkyl.

In a certain embodiment, the bicyclic nucleosides have the formula:

in which:

Bx is a heterocyclic base moiety;

Qa-Qb-Qc- is -CH<2>-N(Rc)-CH<2>-, -C(=O)-N(Rc)-CH<2>-, -CH<2>-O-N( Rc)-, -CH<2>-N(Rc)-O- or - N(Rc)-O-CH<2>;

Rc is C<1>-C<12>alkyl or an amino protecting group; and

Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety or a covalent bond to a supporting medium.

In certain embodiments, the bicyclic nucleosides have the formula:

in which:

Bx is a heterocyclic base moiety;

Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety or a covalent bond to a supporting medium.

Za is C<1>-C<6>alkyl, C<2>-C<6>alkenyl, C<2>-C<6>alkynyl, substituted C<1>-C<6>alkyl, C<alkenyl 2>-C<6>substituted, C<2>-C<6>substituted alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol

In one embodiment, each of the substituted groups is, independently, mono- or polysubstituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJd, SJc, N<3>, OC(=X)Jc, and NJeC(=X)NJcJd, where each Jc, Jd and Je is, independently, H, C<1>-C<6>alkyl, or C<1>-C<6>alkyl and X is O or NJ< c>.

In certain embodiments, the bicyclic nucleosides have the formula:

in which:

Bx is a heterocyclic base moiety;

Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety or a covalent bond to a supporting medium.

Zb is C<1>-C<6>alkyl, C<2>-C<6>alkenyl, C<2>-C<6>alkynyl, substituted C<1>-C<6>alkyl, C<alkenyl 2>-C<6>substituted, C<2>-C<6>substituted alkynyl or substituted (C(=O)-) acyl.

In certain embodiments, the bicyclic nucleosides have the formula:

in which:

Bx is a heterocyclic base moiety;

Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety or a covalent bond to a supporting medium.

Rd is C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C-alkynyl <2>-C<6>substituted C<2>-C<6>alkynyl;

each qa, qb, qc and qd is, independently, H, halogen, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<2>-C6alkenyl, C<2alkenyl >-C<6>substituted, C<2>-C<6>alkynyl or substituted C<2>-C<6>alkynyl, C<1>-C<6>alkoxy, C<1>-C<alkoxy 6>substituted, acyl, substituted acyl, C<1>-C<6>aminoalkyl or substituted C<1>-C<6>aminolakyl;

In certain embodiments, the bicyclic nucleosides have the formula:

in which:

Bx is a heterocyclic base moiety;

Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety or a covalent bond to a supporting medium;

qa, qb, q and qf are each, independently, hydrogen, halogen, C<1>-C<12>alkyl, substituted C<1>-C<12>alkyl, C<2>-C<12>alkenyl, alkenyl C<2>-C<12>substituted, C<2>-C<12>alkynyl, or substituted C<2>-C<12>alkynyl, C<1>-C<1 2>alkoxy, C<alkoxy 1>-C<12>substituted, OJj, SJj, SOJj, SO2Jj, NJjJk, N<3>, CN, C(=O)OJj, C(=O)NJjJk, C(=O)NJjJk, N(H )C(=NH)NJjJk, N(H)C(=O)NJjJk or N(H)C(=S)NJ

or qe and qf together are = C(qg)(qh);

qg and qh are each, independently, H, halogen, C<1>-C<12>alkyl or substituted C<1>-C<12>alkyl.

The synthesis and preparation of bicyclic nucleosides of adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil having a <4>-CH<2>-O-<2>' bridge have been described, together with their oligomerization and properties. nucleic acid recognition ((Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has also been described in WO 98/39352 and WO 99/14226.

Analogs of several bicyclic nucleosides having 4' to 2' bridging groups such as 4'-CH2-O-2' and 4'-<c>H2-S-2' have also been prepared (Kumar et al., Bioorg . Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, the synthesis of 2'-amino-BNA, a new conformationally restricted high-affinity oligonucleotide analogue, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-BNA have been prepared and the thermal stability of their duplexes with RNA strands and has been previously reported.

complementary DNA.

In certain embodiments, the bicyclic nucleosides have the formula:

in which

Bx is a heterocyclic base moiety;

Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugated group, a reactive phosphorus group, a phosphorus moiety or a covalent bond to a supporting medium;

each q i, q , qk and qi is, independently, H, halogen, C<1>-C<12>alkyl, substituted C<1>-C<12>alkyl, C<2>-C<1 2>alkenyl, C<2>-C<12>substituted alkenyl, C<2>-C<1 2>alkynyl, substituted C<2>-C<12>alkynyl, C<1>-C<1 2>alkoxy, C-alkoxy <1>-C<12>substituted, OJj, SJj, SOJj, SO<2>Jj, NJjJk, N<3>, CN, C(=O)OJj, C(=O)NJjJk, C(=O) Jj, OC(=O)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=O)NJjJk or N(H)C(=S)NJjJk; and

qi and q or ql and qk together are =C(qg)(qh), where qg and q h are each, independently, H, halogen, C<1>-C<12>alkyl or C<1>-C<12>alkyl replaced.

A carbocyclic bicyclic nucleoside having a 4'-(CH2)3-2' bridge and the analogous alkenyl bridge 4'-CH=CH-CH2-2' have been described (Frier et al., Nucleic Acids Research, 1997, 25 (22), 4429-4443 and Albaek et al., J. Org., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L-methyleneoxy (4'-CH2-O-2') BNA, (B) p-D-methyleneoxy (4'-CH2-O-2' ) BNA, (C) ethyleneoxy (4'-(CH2)2-O-2') BNA, (D) aminooxy (4'-CH2-O-N(R)-2') BNA, (E) oxyamino (4' -CH2-N(R)-O-2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH3)-O-2') BNA (also called restricted ethyl or cEt), (G) methylene -thio (4-CH2-S-2') b Na, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH (c H3)-2') BNA, (J) propylene carbocyclic (4'-(CH2)3-2') BNA, and (K) vinyl BNA as depicted below.

where Bx is the base moiety and R is, independently, H, a protecting group, C<1>-C<6>alkyl or C<1>-C<6>alkoxy.

As used herein, the term "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted for the pentofuranosyl residue in normal nucleosides and may be referred to as a sugar substitute. Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-<h>N<a>) having a tetrahydropyranyl ring system as illustrated below.

In a certain embodiment, sugar substitutes are selected having the formula:

in which:

Bx is a heterocyclic base moiety;

T<3>and T<4>are each, independently, an internucleoside linking group that links the tetrahydropyran nucleoside analog to the antisense compound or one of T<3>and T<4>is an internucleoside linking group which links the tetrahydropyran nucleoside analog to the antisense compound and the other of T<3>and T<4>is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3' terminal group; q-i, q<2>, q<3>, q<4>, q<5>, q6 and q<7>are each independently, H, C<1>-C<6>alkyl, C<1>alkyl -C<6>substituted, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2>-C<6>alkynyl or C<2>-C<6alkynyl >replaced; and

each of Ri and R<2>is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ<1>J<2>, SJi, N<3>, OC(=X)Ji, OC( =X)NJiJ<2>, NJ<3>C(=X)NJiJ<2>and CN, where X is O, S or NJi and each J-, J<2>and J<3>is, independently , H or Ci-C6 alkyl.

In certain embodiments, qi, q<2>, q<3>, q<4>, q<5>, q6 and q<7> are each H. In certain embodiments, at least one of qi, q<2 >, q<3>, q<4>, q<5>, q6 and q<7> is other than H. In certain embodiments, at least one of qi, q<2>, q<3>, q< 4>, q<5>, q6 and q<7>is methyl. In certain embodiments, THP nucleosides are provided wherein one of Ri and R<2>is F. In certain embodiments, Ri is fluoro and R<2>is H; Ri is methoxy and R<2>is H, and Ri is methoxyethoxy and R<2>is H.

In certain embodiments, the sugar substitutes comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, for example, Braasch et al., Biochemistry, 2002, 4i, 4503-45i0; and US Patents 5,698,685 ; 5.i66.3i5; 5.i85.444; As used herein, the term "morpholino" means a sugar substitute having the following formula:

In certain embodiments, the morpholinos may be modified, for example, by adding or altering various substituent groups of the above morpholino structure. Such sugar substitutes are referred to herein as modified morpholinos.

Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/i0 ii57 published 08/2i/08 for other 5', 2'-nucleosides). disclosed) and the substitution of the oxygen atom of the ribosyl ring with S and the additional substitution at the 2' position (see published United States Patent Application US2005-0i30923, published June 16, 2005) or alternatively 5' substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/i34i8i, published ii/22/07 wherein a bicyclic nucleoside 4'-CH2-O-2- is additionally substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, for example, Srivastava et al., J. Am. Chem. Soc. 2007, i29(26), 8362-8379). .

In certain embodiments, the antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (see, for example, published common property PCT application WO 20i0/036696, published April 10, 2010, Robeyns et al., J. Am . (30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org., 2003, 68, 4499-4505; (24), 4941-4947; Wang et al., J. Org., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785 -788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; PCT application published, WO 06/047842; and PCT Application published WO 01/049687). Certain modified cyclohexenyl nucleosides have the formula X.

wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula

Bx is a heterocyclic base moiety;

T<3>and T<4>are each, independently, an internucleoside linking group that links the cyclohexenyl nucleoside analog to an antisense compound or one of T<3>and T<4>is an internucleoside linking group that links the tetrahydropyran nucleoside analog to the antisense compound and the other of T<3>and T<4>is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3' terminal group; and

q-i, q<2>, q<3>, q<4>, q<5>, q6 and q<7>are each independently, H, C<1>-C<6>alkyl, C<1>alkyl -C<6>substituted, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2>-C<6>alkynyl or C<2>-C<6alkynyl >substituted or other sugar substituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known in the art and are suitable as sugar substitutes that can be used to modify nucleosides for incorporation into oligomeric compounds as provided herein (see, for example, review article: Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo several additional substitutions to further improve their activity.

As used herein, "2'-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to, substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl and substituted and unsubstituted alkynyl. In certain embodiments, the 2' modifications are selected from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH<2>)nNH<2>, O(CH2)nCH3, O( CH<2>)nF, O(CH<2>)nONH<2>, OCH2C(=O)N(H)CH3, and O(CH<2>)nON[(CH<2>)nCH<3> ]<2>, where n and m are from 1 to about 10. Other 2' substituent groups may also be selected from: C<1>-C<12>alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH<3>, OCN, Cl, Br, CN, F, CF<3>, OCF<3>, SOCH<3>, SO<2>CH<3>, ONO<2>, NO<2>, N<3>, NH<2>, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group to improve the pharmacodynamic properties of an antisense compound and other substituents that have similar properties. In certain embodiments, the modified nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such a 2'-MOE substitution has been reported to have improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'-O-methyl, O-propyl and O-aminopropyl. Oligonucleotides having the 2'-MOE substituent have also been shown to be antisense inhibitors of gene expression with promising characteristics for use in vivo (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al. ., Chimia, 1996, 50, 168-176, Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637 and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, "2'-modified" or "2'-substituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH. 2'-modified nucleosides include, but are not limited to, bicyclic nucleosides in which the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with2'-non-bridged substituents, such as allyl, amino, azido, thio, O-allyl, alkyl O-C<1-10>, -OCF<3>, O-(CH<2>)<2>-O- CH<3>, 2'-O(CH2)2SCH3, O-(CH<2>)<2>-O-N(Rm)(Rn), or O-CH<2>-C(=O)-N( Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C<1>-C<10>alkyl. The 2'-modified nucleosides may further comprise other modifications, for example at other positions on the sugar and/or on the nucleobase.

As used herein, "2'-F" refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position of the sugar ring.

As used herein, "2'-OMe" or "<2>-OCH<3>", "2'-O-methyl" or "2'-methoxy" each refers to a nucleoside comprising a sugar comprising a -OCH<3>group at the 2' position of the sugar ring.

As used herein, "MOE" or "2'-MOE" or "<2>-OCH<2>CH<2>OCH<3>" or "2'-O-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a -OCH<2>CH<2>OCH<3> group at the 2' position of the sugar ring.

Methods for preparations of modified sugars are well known to those skilled in the art. Some representative United States patents teaching the preparation of such modified sugars include, without limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed on June 2, 2005 and published as WO 2005/121371 on December 22, 2005.

As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, nucleobase moieties (natural, modified or combination thereof) are retained for hybridization with an appropriate nucleic acid target.

In certain embodiments, the antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the modified 2'-MOE nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a bridging group (4'-CH(CH3)-O-2'). In certain embodiments, modified (4'-CH(CH3)-O-2') nucleosides are arranged along the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, but functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity, or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CEC-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil , 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7 -methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base fractions may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5- propynylcytosine.

In certain embodiments, antisense compounds targeting an ApoCIII nucleic acid comprise one or more modified nucleobases.

RNAi compounds

In certain embodiments, the antisense compounds are RNA interfering (RNAi) compounds, including double-stranded RNA compounds (also called short interfering RNA or siRNA) and single-stranded RNAi (or ssRNA) compounds. Such compounds function at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA compounds/microRNA mimics). In certain embodiments, the antisense compounds comprise modifications that make them particularly suitable for such mechanisms.

Yo. ssRNA compounds

In certain embodiments, antisense compounds including those particularly suitable for use as single-stranded RNAi (ssRNA) compounds comprise a modified 5' terminus. In certain such embodiments, the 5' terminus comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., it is resistant to degradation/cleavage compared to the unmodified 5' phosphate). In certain embodiments, such 5'-terminal nucleosides stabilize the 5'-phosphorose moiety. Certain modified 5'-terminal' nucleosides can be found in the art, for example in WO/2011/139702.

In certain embodiments, the 5' nucleoside of an ssRNA compound has Formula IIC:

in which:

T<1>is an optionally protected phosphorus fraction;

T<2>is an internucleoside linking group that links the compound of Formula IIc to the oligomeric compound;

A has one of the formulas:

Q<1>and Q<2>are each, independently, H, halogen, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<1>-C<alkoxy 6>, substituted C<1>-C<6>alkoxy, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2>-C<6>alkynyl, alkynyl C<2>-C<6>substituted or N(Ra)(R4);

Qa is O, S, N(Ra) or C(R6)(R<t>);

each R<3>, R<4>, R<5>, R6 and R<7>is, independently, H, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl or C<1>-C<6>alkoxy; M<3>is O, S, NR<14>, C(R15)(R16), C(R15)(R16)C(R17)(R18), C(R15)=C(R17), OC(R15 )(R16) or OC(R15)(Bx2);

R<14>is H, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6alkoxy >substituted, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2>-C<6>alkynyl or substituted C<2>-C<6>alkynyl;

R<15>, R<16>, R<17>and R<18>are each, independently, H, halogen, C<1>-C<6>alkyl, C<1>-C<6>alkyl substituted, C<1>-C<6>alkoxy, substituted C<1>-C<6>alkoxy, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C-alkynyl <2>-C<6>or substituted C<1>-C6 alkynyl;

Bx<1>is a heterocyclic base moiety;

or if Bx<2>is present, then Bx<2>is a heterocyclic base moiety and BX<1>is H, halogen, C<1>-C6 alkyl, substituted C<1>-C<6>alkyl, C<1>-C<6>alkoxy, substituted C<1>-C<6>alkoxy, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2alkynyl >-C<6>or substituted C<1>-C<6>alkynyl;

J<4>, J<5>, J6and J<7>are each, independently, H, halogen, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C-alkoxy <1>-C<6>, substituted C<1>-C<6>alkoxy, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2>-alkynyl C<6>, C<2>-C<6>substituted alkynyl;

or J<4>forms a bridge with one of J6 or J<7>wherein said bridge comprises 1 to 3 linked biradical groups selected from O, S, NR<1 9>, C(R<2>o)( R<21>), C(R<2>o)=C(R<21>), C[=C(R<2>o)(R<21>)] and C(=<o>) and the other two of J<5>, J6 and J<7>are each, independently, H, halogen, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C-alkoxy <1>-C<6>, substituted C<1>-C<6>alkoxy, C<2>-C<6>alkenyl, substituted C<2>-C<6>alkenyl, C<2>-alkynyl C<6>, or substituted C<2>-C<6>alkynyl;

each R<19>, R<20>and R<21>is, independently, H, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<1>-alkoxy C<6>, substituted C<1>-C6 alkoxy, C<2>-C<6> alkenyl, substituted C<2>-C<6>alkenyl, C<2>-C<6>alkynyl, or alkynyl C<2>-C<6>substituted;

G is H, OH, halogen or O-[C(R<8>)(R<9>)]n[(C=O)m-x<1>]j-Z;

each R8 and R<9>is, independently, H, halogen, C<1>-C<6>alkyl or substituted C<1>-C<6>alkyl;

X<1>is O, S or N(E<1>);

Z is H, halogen, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<2>-C<6>alkenyl, C<2>-C<6>alkenyl substituted, C<2>-C<6>alkynyl, substituted C<1>-C<6>alkynyl or N(E<2>)(E<3>);

E<1>, E<2>and E<3>are each, independently, H, C<1>-C<6>alkyl or substituted C<1>-C<6>alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ<1>, N(J<1>)(J<2>), =NJ<1>, SJ<1>, N<3> , CN, OC(=X<2>)J<1>, OC(=X<2>)-N(J<1>)(J<2>) and C(=X2)N(J1)(J2 );

X<2>is O, S or NJ<3>;

each J<1>, J<2>and J<3>is, independently, H or C<1>-C<6>alkyl;

when j is 1 then Z is other than halogen or N(E<2>)(E<3>); and

wherein said oligomeric compound comprises 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

In certain embodiments, M<3>is O, CH=CH, OCH<2>or OC(H)(Bx<2>). In certain embodiments M<3>is O.

In certain embodiments, J<4>, J<5>, J6 and J<7> are each H. In certain embodiments, J<4> forms a bridge with one of J<5>or J<7>.

In certain embodiments, A has one of the formulas:

in which:

Q<1>and Q<2>are each, independently, H, halogen, C<1>-C<6>alkyl, substituted C<1>-C<6>alkyl, C<1>-C<alkoxy 6>, substituted C<1>-C<6>alkoxy. In certain embodiments, Q<1>and Q<2>are each H. In certain embodiments, Q<1>and Q<2>are each, independently, H or halogen. In certain embodiments, Q<1>and Q<2>is H and the other of Q<1>and Q<2>is F, CH<3>or OCH<3>.

In certain embodiments, T<1> has the formula:

in which:

Ra and Rc are each, independently, protected hydroxyl, protected thiol, C<1>-C<6>, substituted C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C-alkoxy <1>-C<6>substituted, protected amino or substituted amino; and

Rb is O or S. In certain embodiments, Rb is O and Ra and Rc are each, independently, OCH<3>, OCH<2>CH<3>or CH(CH3)2.

In certain embodiments, G is halogen, OCH<3>, OCH<2>F, OCHF<2>, OCF<3>, OCH<2>CH<3>, O(CH<2>)<2>F, OCH<2>CHF<2>, OCH<2>CF<3>, OCH<2>-CH=CH<2>, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2) 2-OCF3, O(CH2)3-N(R10)(Rh ), O(CH2)2-ON(R<10>)(R<11>), O(CH<2>)<2>-O (CH<2>)<2>-N(R<10>)(R<11>), OCH<2>C(=O)-N(R<10>)(R<11>), OCH< 2>C(=O)-N(R<12>)-(CH<2>)<2>-N(R<10>)(R<11>) or O(CH<2>)<2> -N(R<12>)-C(=NR<13>)[N(R<10>)(Rh )] where R<10>, R<11>, R<12>and R<13> They are each, independently, H or C<1>-C<6>alkyl. In certain embodiments, G is halogen, OCH<3>, OCF<3>, OCH<2>CH<3>, OCH<2>CF<3>, OCH<2>-CH=CH<2>, O( CH<2>)<2>-OCH<3>, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)- N(H)-(CH2)2-N(CH3)2 or OCH<2>-N(H)-C(=NH)NH<2>. In certain embodiments, G is F, OCH<3>or O(CH<2>)<2>-OCH<3>. In certain embodiments, G is O(CH<2>)<2>-OCH<3>.

In certain embodiments, the 5'-terminal nucleoside has the formula lie:

In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more types of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or motif. sugar modification. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2'-F nucleoside. In certain embodiments, each nucleoside of the region is a 2'-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, the oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, the nucleosides of both types are RNA-type nucleosides. In certain embodiments, the alternate nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA and cEt. In certain embodiments, the alternate modifications are 2'-F and 2'-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consists of a single nucleoside (i.e., the pattern is (AB)xAy, where A is a nucleoside having a sugar modification of a first type and B is a nucleoside having has a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternate regions in an alternate motif include more than a single nucleoside of one type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; either

ABBBAABBABABAA;

where A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2'-F, 2'-OMe, BNA and MOE.

In certain embodiments, oligonucleotides having such an alternate motif also comprise a modified 5'-terminal nucleoside, such as those of formula IIC or IIe.

In certain embodiments, the oligonucleotides comprise a region having a 2-2-3 motif. These regions include the following reason:

-(A)2-(B)x-(A)2-(C)y-(A)3

where: A is a first type of modified nucleoside;

B and C are nucleosides that are modified differently than A, however, B and C can have the same or different modifications from each other;

x and y are 1 to 15.

In certain embodiments, A is a 2'-OMe modified nucleoside. In certain embodiments, B and C are 2'-F modified nucleosides. In certain embodiments, A is a 2'-OMe modified nucleoside and B and C are both 2'-F modified nucleosides.

In certain embodiments, the oligonucleosides have the following sugar motif:

5'- (Q)- (AB)xAy-(D)z

where:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a different modification than the nucleoside adjacent to it. Therefore, if y is 0, then D must be modified differently than B and if y is 1, then D must be modified differently than A. In certain embodiments, D is different from both A and B.

X is 5-15;

And it is 0 or 1;

Z is 0-4.

In certain embodiments, the oligonucleosides have the following sugar motif:

5'-(Q)-(A)x-(D)z

where:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula lIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a different modification of A.

X is 11-30;

Z is 0-4.

In certain embodiments, A, B, C and D in the above motifs are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, said terminal nucleosides are not designed to hybridize with the target nucleic acid (although one or more could hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments, the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, including those particularly suitable for use as ssRNA comprise modified internucleoside bonds arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside bond motif. In certain embodiments, the oligonucleotides comprise a region having an alternating internucleoside binding motif. In certain embodiments, the oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside bonds. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside bonds. In certain embodiments, each internucleoside bond of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least 12 consecutive phosphorothioate internucleoside bonds. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein may have any linker motif. For example, oligonucleotides, including but not limited to those described above, may have a binding motif selected from the following non-limiting table:

ii. siRNA compounds

In certain embodiments, the antisense compounds are double-stranded RNAi (siRNA) compounds. In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, the ssRNA compounds may be unmodified RNA. In certain embodiments, the siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

Various embodiments relate to double-stranded compositions wherein each chain comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided that comprise a first and a second oligomeric compound that are completely or at least partially hybridized to form a duplex region and that further comprise a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that said composition comprises a first oligomeric compound that is an antisense chain that has full or partial complementarity with a nucleic acid target and a second oligomeric compound that is a sense chain that has one or more regions of complementarity with and forms by at least one duplex region with the first oligomeric compound.

Compositions of various embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is ApoCIII. In certain embodiments, targeted ApoCIII degradation is facilitated by an activated RISC complex that is formed with the compositions of the invention.

Various embodiments are directed to double-chain compositions in which one of the chains is useful in, for example, influencing the preferential charge of the opposite chain in the RISC complex (or cleavage). The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

Certain embodiments focus on dual-chain compositions where both chains comprise a hemimer motif, a fully modified motif, a positionally modified motif, or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfill a particular role in, for example, the siRNA pathway. Using a different motif on each strand or the same motif with different chemical modifications on each strand allows targeting the antisense strand to the RISC complex while inhibiting incorporation of the sense strand. Within this model, each chain can be modified independently so that it is improved for its particular function. The antisense strand can be modified at the 5' end to improve its function in one region of the RISC while the 3' end can be differentially modified to improve its function in a different region of the RISC.

The double-stranded oligonucleotide molecules may be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to the nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, where the sense and antisense strands are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence on the other strand; such as when the antisense strand and the sense strand form a duplex or double-stranded structure, for example, where the double-stranded region is approximately 15 a about 30, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs comprise the nucleotide sequence; that is complementary to the nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked via a nucleic acid-based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide may be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to the nucleotide sequence. in a separate target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide may be a circular single-stranded polynucleotide having two or more turn structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises the nucleotide sequence that is complementary to the sequence of nucleotides in a target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules as is known in the art, or alternatively are linked. non-covalently through ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises a nucleotide sequence that is complementary to the nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with the nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but also encompass chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2'-hydroxy (2'-OH)-containing nucleotides. In certain embodiments, the short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support the RNAi may however have an attached linker or linkers or other attached or associated groups, moieties or chains that contain one or more nucleotides with 2'-groups. OH. Optionally, double-stranded oligonucleotides may comprise ribonucleotides at approximately 5, 10, 20, 30, 40 or 50% of the nucleotide positions. As used herein, the term siRNA is intended to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence-specific RNAi, e.g., short interfering RNA (siRNA, double-stranded RNA ( dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgRNA), ssRNA and others. As used herein, the term RNAi is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, or epigenetics. For example, stranded oligonucleotides may be used. double to silence genes epigenetically at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention may be the result of siRNA-mediated modification of the gene. chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

Compounds and compositions for use in the invention may target ApoCIII via a dsRNA-mediated gene silencing or RNAi mechanism, including, for example, "hairpin" or stem-turn double-stranded RNA effector molecules in which a Single-stranded RNA with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed April 19, 2000, or U.S. Pat. Serial No. 60/130,377, filed April 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in a base segment of the molecule pair with nucleotides. in another segment of the molecule. In various embodiments, a dsRNA consisting of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity with each other.

In various embodiments, both chains consist entirely of ribonucleotides, one chain consists entirely of ribonucleotides and one chain consists entirely of deoxyribonucleotides, or one or both chains contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the complementarity regions are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100,200 , 500, 1000, 2000 or 5000 nucleotides or includes all nucleotides in a cDNA or other target nucleic acid sequence represented in the dsRNA. In some embodiments, the dsRNA does not contain any single-stranded regions, such as single-strand ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single-stranded or overhang regions. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g., has at least 70, 80, 90, 95, 98, or 100% complementarity with an acid). target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g., has at least 70, 80, 90, 95, 98, or 100% identity with a target nucleic acid), and vice versa .

In various embodiments, the RNA/DNA hybrid is prepared in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed April 19, 2000, or U.S. Pat. Serial No. 60/130,377, filed April 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after or concurrently with the transformation of the DNA chain in the cell. In still other embodiments, the dsRNA is a single circular nucleic acid containing a sense and antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00 /63364, filed April 19, 2000, or U.S. Serial No. 60/130,377, filed April 21, 1999. Exemplary nucleic acids include loop structures in which the free 5' phosphoryl group of a nucleotide binds to the 2' hydroxyl group of another nucleotide in a turn-around manner.

In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as a fluorine group) or contains an alkoxy group (such as a methoxy group) that increases the half-life of the dsRNA. in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2' position contains a hydrogen or a hydroxyl group. In still other embodiments, the dsRNA includes one or more bonds between adjacent nucleotides other than a naturally occurring phosphodiester bond. Examples of such bonds include phosphoramide, phosphorothioate and phosphorodithioate bonds. dsRNAs can also be chemically modified nucleic acid molecules as described in US Patent No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed April 19, 2000, or U.S. Pat. Serial No. 60/130,377, filed on April 21, 1999.

In other embodiments, the dsRNA may be any of the dsRNA molecules at least partially disclosed in WO 00/63364, as well as any of the dsRNA molecules described in US Provisional Application 60/399,998; and US Provisional Application 60/419,532, and PCT/US2003/033466. Any of the dsRNAs can be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Compositions and Methods for Formulating Pharmaceutical Compositions

The antisense compounds may be mixed with a pharmaceutically acceptable active or inert substance for the preparation of pharmaceutical compositions or formulations. Compositions and methods for formulating pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

Antisense compounds targeting an ApoCIII nucleic acid can be used in pharmaceutical compositions by combining the antisense compound with a pharmaceutically acceptable diluent or carrier.

In certain embodiments, the "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and may be selected, with the planned mode of administration in mind, to provide volume, consistency, etc. desired, when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.); fillers (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.) lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (for example, starch, sodium starch glycolate, etc.); and wetting agents (e.g. sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with nucleic acids, suitable for parenteral or non-parenteral administration, may also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

A pharmaceutically acceptable diluent includes phosphate buffered saline (PBS). PBS is a suitable diluent for use in compositions that are administered parenterally. Accordingly, in one embodiment, a pharmaceutical composition comprising an antisense compound targeting an ApoCIII nucleic acid and a pharmaceutically acceptable diluent is employed in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salt, ester or salt of said ester, or any other oligonucleotide that, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the metabolite or residue. of the same biologically active. Accordingly, for example, the disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug may include the incorporation of additional nucleosides at one or both ends of an antisense compound that are cleaved by endogenous nucleases within the body to form the active antisense compound.

Conjugated Antisense Compounds

The antisense compounds can be covalently linked to one or more moieties or conjugates that improve the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugated groups include cholesterol moieties and lipid moieties. Additional conjugated groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that generally attach to one or both terminal ends of antisense compounds to improve properties such as nuclease stability. Included in the stabilizing groups are the cap structures. These terminal modifications protect the antisense compound from degradation by exonucleases, and may aid delivery and/or localization within a cell. The cap may be present at the terminal end 5' (cap 5), or at the terminal end 3' (cap 3'), or may be present at both terminal ends. Cap structures are well known in the art and include, for example, inverted deoxybasic caps. Additional 3' and 5' stabilizing groups that can be used to cap one or both ends of an antisense compound to impart stability to nucleases include those disclosed in WO 03/004602 published January 16, 2003.

Cell Culture and Treatment with Antisense Compounds

The effects of antisense compounds on the level, activity or expression of ApoCIII nucleic acids can be tested in vitro in a variety of cell types. The cell types used for such analyzes are available from commercial vendors (e.g., American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and the cells were Cultured according to the seller's instructions using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells.

In vitro tests of Antisense Oligonucleotides

Described herein are methods for treating cells with antisense oligonucleotides, which can be appropriately modified for treatment with other antisense compounds.

In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

A commonly used reagent to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, CA). The antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of the antisense oligonucleotide and a LIPOFECTIN® concentration that typically ranges from 2 to 12 ug/ml per 100 nM of antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, CA). The antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a concentration of LIPOFECTAMINE® typically ranging from 2 to 12 ug/ml per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, CA). The antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a concentration of Cytofectin® typically ranging from 2 to 12 ug/ml per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Oligofectamine™ (Invitrogen Life Technologies, Carlsbad, CA). The antisense oligonucleotide is mixed with Oligofectamine™ in Opti-MEM™-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of oligonucleotide with a ratio of Oligofectamine™ to oligonucleotide of approximately 0.2 to 0 .8 pl per 100 nM.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis, IN). The antisense oligomeric compound was mixed with FuGENE 6 in 1 ml of serum-free RPMI to achieve the desired concentration of oligonucleotide with a ratio of FuGENE 6 to oligomeric compound of 1 to 4 μl of FuGENE 6 per 100 nM.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation (Sambrook and Russell, in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001).

Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after treatment with antisense oligonucleotides, at which time RNA or protein levels of the target nucleic acids are measured by methods known in the art and described herein (Sambrook and Russell, in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. In general, when treatments are performed in multiple replicates, data are presented as the mean of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods for determining the optimal concentration of antisense oligonucleotides for a particular cell line are well known in the art (Sambrook and Russell, in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000® (Invitrogen, Carlsbad, CA), Lipofectin (Invitrogen, Carlsbad, CA), or Cytofectin™ (Genlantis, San Diego, CA) .. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art (Sambrook and Russell, in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). RNA is prepared using methods well known in the art, for example, using TRIZOL® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.

Analysis of Inhibition Levels or Target Expression

Inhibition of the levels or expression of an ApoCIII nucleic acid can be assayed in a variety of ways known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). For example, target nucleic acid levels can be quantified by, for example, Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time quantitative PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently carried out using the commercially available ABI PRISM® 7600, 7700 or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to the manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantification of target RNA levels can be achieved by real-time quantitative PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Quantitative real-time PCR methods are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). The RT, and real-time PCR reactions are carried out by methods well known to those skilled in the art.

Target amounts of genes (or RNA) obtained by real-time PCR can be normalized by using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGRE<e>N® ( Invitrogen, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real-time PCR, running simultaneously with the target, by multiplexing, or separately. Total RNA is quantified using RIBOGREEN® RNA Quantification Reagent (Invitrogen, Inc. Carlsbad, CA). RNA quantification methods using RIBOGREEN® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems, Foster City, CA) is used to measure RIBOGREEN® fluorescence.

The probes and primers are designed to hybridize with an ApoCIII nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS® software (Applied Biosystems, Foster City, CA).

Gene target quantities obtained by RT, real-time PCR can use either the expression level of GAPDH or Cyclophilin A, genes whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, OR). The expression of GAPDH or Cyclophilin A can be quantified by RT, real-time PCR, running simultaneously with the target, by multiplexing or separately. Total RNA was quantified using RiboGreen™ RNA Quantification Reagent (Molecular Probes, Inc. Eugene, OR).

Analysis of Protein Levels

Antisense inhibition of ApoCIII nucleic acids can be assessed by measuring ApoCIII protein levels. ApoCIII protein levels can be assessed or quantified in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays. proteins (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS) (Sambrook and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001).) Targeted antibodies can be identified and obtained from a variety of sources, such as the MSRS antibody catalog (Aerie Corporation, Birmingham, MI), or can be prepared by monoclonal or polyclonal antibody generation methods. conventional ones well known in the art. Antibodies useful for the detection of human and mouse ApoCIII are commercially available.

In vivo testing of antisense compounds

Antisense compounds, for example, antisense oligonucleotides, are tested in animals for their ability to inhibit ApoCIII expression and produce phenotypic changes. Tests can be performed on normal animals or on experimental disease models. For administration to animals, the antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration. Calculation of antisense oligonucleotide dosage and dosing frequency depends on factors such as the route of administration and the body weight of the animal. After a period of treatment with antisense oligonucleotides, RNA is isolated from the tissue and changes in ApoCIII nucleic acid expression are measured. Changes in ApoCIII protein levels are also measured.

Certain Indications

The novel effects of ApoCIII inhibition in patients with Fredrickson dyslipidemia Type I, FCS, LPLD have been identified and reported herein. The example disclosed herein below discloses striking reductions in TG and increases in HDL among other biomarkers in patients with Fredrickson Dyslipidemia Type I, FCS, LPLD who have little or no detectable LPL activity.

Without being bound by any particular theory, two possible explanations for the surprising results are discussed. First, inhibition of ApoCIII may activate residual LPL activity in patients with Fredrickson dyslipidemia Type I, FCS, LPLD. This is not a very likely explanation since these patients have little or no detectable LPL activity, while ApoCIII inhibition has profoundly affected TG and HDL levels. Second, and more likely, ApoCIII inhibits the clearance of TG particles mediated by apoE-mediated receptors, such as low-density lipoprotein receptor-related protein 1 (LRP1) or Syndecan 1. Once cleared ApoCIII from VLDL particles and chylomicrons become easier to be taken up by the liver. Indeed, these receptor-mediated clearance mechanisms may contribute significantly to the clinically observed phenotype (e.g., substantial TG decrease) observed in patients with Fredrickson dyslipidemia Type I, FCS, LPLD, treated with an ApoCIII inhibitor.

Disclosed herein are methods for treating a subject with Fredrickson Dyslipidemia Type I, FCS, LPLD, comprising the administration of one or more pharmaceutical compositions as described herein. In certain embodiments, the pharmaceutical composition comprises an antisense compound targeting an ApoCIII.

In certain embodiments, administration of an antisense compound targeting an ApoCIII nucleic acid to a subject with Fredrickson dyslipidemia Type I, FCS, LPLD, results in reduction of ApoCIII expression by at least about 15, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, ApoCIII expression is reduced to <50 mg/L, <60 mg/L, <70 mg/L, <80 mg/L, <90 mg/L, <100 mg/L, <110 mg /l, < 120 mg/l, < 130 mg/l, < 140 mg/l, < 150 mg/l, < 160 mg/l, < 170 mg/l, < 180 mg/l, < 190 mg/l or < 200 mg/l.

In certain embodiments, the subject has a disease or disorder related to Fredrickson dyslipidemia Type I, FCS, LPLD. In certain embodiments, the disease or disorder is a cardiovascular or metabolic disease or disorder. In certain embodiments, the disease is pancreatitis.

In certain embodiments, cardiovascular disease includes, but is not limited to, aneurysm, angina pectoris, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, stroke, and the like. In certain embodiments, the dyslipidemia is chylomicronemia (e.g., FCS) or hypertriglyceridemia. In certain embodiments, the disease is pancreatitis caused by dyslipidemia.

In certain embodiments, the metabolic disease or disorder includes, but is not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome, and diabetic dyslipidemia.

In certain embodiments, compounds targeting ApoCIII as described herein modulate physiological markers or phenotypes of pancreatitis, a cardiovascular or metabolic disease or disorder in a subject with Fredrickson dyslipidemia Type I, FCS, LPLD. In certain experiments, compounds may increase or decrease physiological markers or phenotypes compared to untreated animals. In certain embodiments, the increase or decrease in physiological markers or phenotypes is associated with the inhibition of ApoCIII by the compounds described herein.

In certain embodiments, the physiological markers or phenotype of a cardiovascular disease or disorder may be quantifiable. For example, TG or HDL levels can be measured and quantified using, for example, standard lipid tests. In certain embodiments, physiological markers or phenotypes such as HDL can be increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, or 99%, or an interval defined by any two of these values. In certain embodiments, the phenotypes of physiological markers such as TG (postprandial or fasting) can be reduced by approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or an interval defined by any two of these values. In certain embodiments, the TG (postprandial or fasting) is reduced to <100 mg/dl, <110 mg/dl, <120 mg/dl, <130 mg/dl, <140 mg/dl, <150 mg/dl , < 160 mg/dl, < 170 mg/dl, < 180 mg/dl, < 190 mg/dl, < 200 mg/dl, < 210 mg/dl, < 220 mg/dl, < 230 mg/dl, < 240 mg/dl, < 250 mg/dl, < 260 mg/dl, < 270 mg/dl, < 280 mg/dl, < 290 mg/dl, < 300 mg/dl, < 350 mg/dl, < 400 mg /dl, < 450 mg/dl, < 500 mg/dl, < 550 mg/dl, < 600 mg/dl, < 650 mg/dl, < 700 mg/dl, < 750 mg/dl, < 800 mg/dl , < 850 mg/dl, < 900 mg/dl, < 950 mg/dl, < 1000 mg/dl, < 1100 mg/dl, < 1200 mg/dl, < 1300 mg/dl, < 1400 mg/dl, < 1500 mg/dl, < 1600 mg/dl, < 1700 mg/dl, < 1800 mg/dl or < 1900 mg/dl.

In certain embodiments, physiological markers or phenotypes of a metabolic disease or disorder may be quantifiable. For example, glucose levels or insulin resistance can be measured and quantified using standard assays known in the art. In certain embodiments, physiological markers or phenotypes such as glucose levels or insulin resistance may be reduced by approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 95 or 99%, or an interval defined by any two of these values. In certain embodiments, the phenotypes of physiological markers such as insulin sensitivity can be increased by approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or an interval defined by any two of these values.

Also disclosed herein are methods of preventing, treating or ameliorating a symptom associated with a disease or disorder in a subject with Fredrickson Dyslipidemia Type I, FCS, LPLD with a compound described herein. Disclosed herein is a method for reducing the rate of occurrence of a symptom associated with a disease associated with Fredrickson Dyslipidemia Type I, FCS, LPLD. Disclosed herein is a method of reducing the severity of a symptom associated with Fredrickson dyslipidemia Type I, FCS, LPLD. In such embodiments, the methods comprise administering to an individual with Fredrickson Dyslipidemia Type I a therapeutically effective amount of a compound targeting an ApoCIII nucleic acid. In certain embodiments, the disease or disorder is pancreatitis or a cardiovascular or metabolic disease or disorder.

Cardiovascular diseases or disorders are characterized by numerous physical symptoms. Any symptom known to one skilled in the art to be associated with cardiovascular disease may be prevented, treated, ameliorated or otherwise modulated, as set forth in the methods described herein. In certain embodiments, the symptom may be any of, but not limited to, angina pectoris, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the extremities, claudication or muscle cramps, swelling of the abdomen or fever.

Metabolic diseases or disorders are characterized by numerous physical symptoms. Any symptom known to one skilled in the art to be associated with a metabolic disorder may be prevented, treated, ameliorated or otherwise modulated as set forth in the methods described herein. In certain embodiments, the symptom may be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.

Pancreatitis is characterized by numerous physical symptoms. Any symptoms known to one skilled in the art to be associated with pancreatitis may be prevented, treated, ameliorated or otherwise modulated as set forth in the methods described herein. In certain embodiments, the symptom may be any of, but not limited to, abdominal pain, vomiting, nausea, and abdominal pressure sensitivity.

Disclosed herein are methods of treating a subject with Fredrickson's dyslipidemia Type I, FCS, LPLD, comprising administering a therapeutically effective amount of one or more pharmaceutical compositions as described herein. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeting an ApoCIII nucleic acid is accompanied by monitoring ApoCIII levels or disease markers associated with Fredrickson Type I dyslipidemia, FCS, LPLD, to determine a subject's response to the antisense compound. A subject's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeting ApoCIII are used for the preparation of a medicament for treating a subject with Fredrickson's dyslipidemia Type I, FCS, LPLD.

Administration

The compounds or pharmaceutical compositions of the present invention can be administered in various ways depending on whether local or systemic treatment is desired and the area to be treated. Administration can be oral or parenteral.

In certain embodiments, the compounds and compositions as described herein are administered parenterally. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion.

In certain embodiments, parenteral administration is by infusion. The infusion can be chronic or continuous or short or intermittent. In certain embodiments, the infused pharmaceutical agents are administered with a pump. In certain embodiments, the infusion is intravenous.

In certain embodiments, parenteral administration is by injection. The injection can be given with a syringe or pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or organ. In certain embodiments, parenteral administration is subcutaneous.

In certain embodiments, formulations for parenteral administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

In certain embodiments, formulations for oral administration of the compounds or compositions of the invention may include, but are not limited to, pharmaceutical carriers, excipients, powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules. , gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In certain embodiments, oral formulations are those in which the compounds of the invention are administered together with one or more penetration enhancers, surfactants and chelators.

Dosage

In certain embodiments, the pharmaceutical compositions are administered according to a dosage regimen (e.g., dosage, dosing frequency and duration) wherein the dosage regimen can be selected to achieve a desired effect. The desired effect may be, for example, reduction of ApoCIII or the prevention, reduction, improvement or slowing of the progression of a disease or condition associated with Fredrickson dyslipidemia Type I, FCS, LPLD.

In certain embodiments, the dosage regimen variables are adjusted to result in a desired concentration of pharmaceutical composition in a subject. "Concentration of pharmaceutical composition as used in connection with the dosage regimen may refer to the compound, oligonucleotide or active ingredient of the pharmaceutical composition. For example, in certain embodiments, the dose and dosing frequency are adjusted to provide a concentration of tissue or plasma concentration of a pharmaceutical composition in an amount sufficient to achieve a desired effect.

The dosage depends on the severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a decrease in the disease state is achieved. . Dosage also depends on the drug's potency and metabolism. In certain embodiments, the dosage is 0.01 mg to 100 mg per kg of body weight, or within a dosage range of 0.001 mg - 1000 mg, and may be administered once or more daily, weekly, monthly or annually. , or even once every 2 to 20 years. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 mg to 100 mg per dose. kg body weight, one or more times a day, once every 20 years or varying from 0.01 mg to 1000 mg dosage.

Certain Combination Therapies

In certain embodiments, a first agent comprising the compound described herein is coadministered with one or more secondary agents. In certain embodiments, such second agents are designed to treat the same disease, disorder or condition as the first agent described herein. In certain embodiments, such second agents are designed to treat a disease, disorder or condition other than that of the first agent described herein. In certain embodiments, a first agent is designed to treat an unwanted side effect of a second agent. In certain embodiments, the second agents are coadministered with the first agent to treat an undesirable effect of the first agent. In certain embodiments, such second agents are designed to treat an unwanted side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, the second agents are coadministered with the first agent to produce a combinatorial effect. In certain embodiments, the second agents are coadministered with the first agent to produce a synergistic effect. In certain embodiments, coadministration of the first and second agents allows the use of lower dosages than would be required to achieve a therapeutic or prophylactic effect if the agents were administered as independent therapy. In certain embodiments, the first agent is administered to a subject who has failed or has become unresponsive to a second agent. In certain embodiments, the first agent is administered to a subject in replacement of a second agent.

In certain embodiments, one or more compositions described herein and one or more different pharmaceutical agents are administered at the same time. In certain embodiments, one or more compositions of the invention and one or more pharmaceutical agents are administered at different times. In certain embodiments, one or more compositions described herein and one or more different pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more compositions described herein and one or more different pharmaceutical agents are prepared separately.

In certain embodiments, the second agents include, but are not limited to, ApoCIII lowering agent, DGAT1 inhibitor, LPL raising agent, cholesterol lowering agent, non-HDL lipid (e.g., LDL) lowering agent, HDL elevation, fish oil, niacin (nicotinic acid), fibrate, statin, DCCR (diazoxide salt), glucose lowering agent and/or antidiabetic agents. In certain embodiments, the first agent is administered in combination with the maximum tolerated dose of the second agent. In certain embodiments, the first agent is administered to a subject who does not respond to a maximum tolerated dose of the second agent.

Examples of ApoCIII reducing agents include an ApoCIII antisense oligonucleotide different from the first agent, fibrate, or an Apo B antisense oligonucleotide.

An example of a DGAT1 inhibitor is LCQ908 (Novartis Pharmaceuticals) which is currently being tested in a Phase 3 clinical trial to treat Familial Chylomicronemia Syndrome (FCS).

LPL-raising agents include gene therapy agents that raise the level of LPL. Examples of such agents include copies of normal genes that supplement the lack of the normal gene. For example, GlyberaR raises LPL levels by providing normal copies of the LPL gene to supplement a lack of the normal LPL gene. In other examples, the LPL-elevating agent includes normal copies of ApoC-II, GPIHBP1, APOA5, LMF1 or other genes that, when mutated, can lead to dysfunctional LPL. In certain embodiments, the combination of the first agent (e.g., ApoCIII ASO) and the second agent (e.g., Glybera) provides an additive or synergistic effect. In certain embodiments, the first agent (e.g., ApoCIII ASO) is administered to a subject who has failed or no longer responds to a second agent (e.g., GlyberaR).

Examples of glucose-lowering and/or antidiabetic agents include, but are not limited to, a therapeutic lifestyle change, PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or a insulin analog, an insulin secretagogue, an SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, metformin, sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione, alpha-glucosidase inhibitor and the like. The sulfonylurea may be acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide or a gliclazide. Meglitinide can be nateglinide or repaglinide. The thiazolidinedione can be pioglitazone or rosiglitazone. Alpha-glucosidase can be acarbose or miglitol.

Cholesterol or lipid-lowering therapy may include, but is not limited to, therapeutic lifestyle change, statins, bile acid sequestrants, nicotinic acid, and fibrates. Statins may be atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin and the like. Bile acid sequestrants may be colesevelam, cholestyramine, colestipol and the like. Fibrates can be gemfibrozil, fenofibrate, clofibrate and the like. Therapeutic lifestyle change may be dietary fat restriction.

HDL-raising agents include cholesteryl ester transfer protein (CETP) inhibitor drugs (such as Torcetrapib), peroxisome proliferator-activated receptor agonists, Apo-A1, Pioglitazone, and the like.

Certain Treatment Populations

Some types of hypertriglyceridemia can be characterized by the Fredrickson classification system or by the classification system described by Tremblay (Tremblay et al., J Clin Lipidol, 2011, 5:37-44). In certain embodiments, the compounds, compositions and methods described herein are useful for treating subjects with Fredrickson's dyslipidemia Type I, FCS, LPLD.

Subjects with Fredrickson dyslipidemia Type I, FCS, LPLD, have a significant risk of pancreatitis, cardiovascular and metabolic disease. For these subjects, recurrent pancreatitis is the most debilitating and potentially lethal complication; Other sequelae include an increased tendency toward atherosclerosis and diabetes.

Subjects with Fredrickson type I, FCS, LPLD, lack a significant amount of functionally active LPL. ApoCIII plays an important role in TG metabolism and is an independent risk factor for cardiovascular disease in subjects with functional or partially functional LPL. ApoCIII is currently in clinical trials to treat subjects with non-Fredrickson type I hypertriglyceridemia. However, as the ApoCIII pathway is thought to function through the LPL pathway, ApoCIII inhibition has not been considered as a treatment option for Fredrickson Type I, FCS, LPLD subjects.

Inhibition of ApoCIII, as shown herein, unexpectedly decreases TG levels and/or elevates HDL levels in subjects with Fredrickson dyslipidemia Type I, FCS, LPLD. Decreasing TG and/or increasing HDL can, in turn, prevent, treat, delay or improve a disease, disorder or symptom thereof, associated with Fredrickson Type I dyslipidemia, FCS, LPLD.

Certain Compounds

We have previously disclosed compositions comprising antisense compounds targeting ApoCIII and methods for inhibiting ApoCIII by the antisense compounds in US 20040208856 (US Patent 7,598,227), US 20060264395 (US Patent 7,750,141), WO 2004/ 093783 and WO 2012/149495. In these applications, a series of antisense compounds were designed to target different regions of the human ApoCIII RNA, using published sequences (nucleotides 6238608 to 6242565 of GenBank accession no. NT_035088.1, which represents a genomic sequence, incorporated into the present as SEQ ID NO: 4, and GenBank Accession No. NM 000040.1, incorporated herein as SeQ ID NO: 1). The compounds were chimeric oligonucleotides ("gapmers") of 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by " "wings" of five nucleotides. The wings are composed of 2'-O-(2-methoxyethyl) nucleotides, also known as (2'-MOE) nucleotides. The internucleoside bonds (main chain) are phosphorothioate (P=S) throughout the oligonucleotide. All cytosine residues are 5-methylcytosines.

Antisense compounds were analyzed for their effect on human ApoCIII mRNA levels in HepG2 cells by real-time quantitative PCR. Several compounds demonstrated at least 45% inhibition of ApoCIII mRNA and are therefore preferred. Several compounds demonstrated at least 50% inhibition of human ApoCIII mRNA and are therefore preferred. Several compounds demonstrated at least 60% inhibition of human ApoCIII mRNA and are therefore preferred. Several compounds demonstrated at least 70% inhibition of human ApoCIII mRNA and are therefore preferred. Several compounds demonstrated at least 80% inhibition of human ApoCIII mRNA and are therefore preferred. Several compounds demonstrated at least 90% inhibition of human ApoCIII mRNA and are therefore preferred.

The target regions to which these preferred antisense compounds are complementary are referred to as "preferred target segments" and are therefore preferred to be targeted by antisense compounds.

EXAMPLES

Example 1: ISIS 304801 Clinical Trial

As described herein, an open-label study was conducted in patients with Fredrickson Dyslipidemia Type I, FCS, LPLD, to evaluate the response to, and pharmacodynamic effects of, the ISIS 304801 Drug Study. ISIS 304801 had been previously disclosed in US Patent 7,598,227 and has the sequence 5'-AGCTTCTTGTCCAGCTTTAT-3' (SEQ ID NO: 3) beginning at position 508 in SEQ ID NO: 1 (GENBANK Accession No. NM_000040.1) or starting at position 3139 in SEQ ID NO: 2 (GENBANK Accession No. NT_033899.8 truncated from nucleotides 20262640 to 20266603). ISIS 304801 has a 5-10-5 MOE gapmer motif comprising a gap segment consisting of 10 linked deoxynucleosides, a 5' wing segment consisting of 5 linked nucleosides, a 3' wing segment consisting of 5 nucleosides linked, wherein the gap segment is positioned immediately adjacent to and between the 5' wing segment and 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methioxyethyl sugar, wherein each cytosine is a 5'-methylcytosine, and where each internucleoside bond is a phosphorothioate bond. ISIS 304801 has been shown to be potent in inhibiting ApoC-III and tolerable when administered to subjects.

Many of the patients recruited for this study had been diagnosed with Fredrickson dyslipidemia Type I, FCS, LPLD. Patients with Fredrickson Type I, FCS, LPLD, with a history of TG levels >880mg/dl, fasting TG levels 750mg/dl during the study examination and/or TG level >440mg/dl were included in the study. dl after the diet but before the start of treatment.

To expand the study population, some patients who have hyperTG but have not been diagnosed with Fredrickson dyslipidemia Type I, FCS, LPLD, can be screened for Fredrickson dyslipidemia Type I, FCS, LPLD. In one example, patients with hyperTG will be identified through their medical history with a TG level of >880 mg/dL and/or by centrifugation of lipids in their blood for a fasting TG level of >750 mg/dL. dl. Patients with a fasting TG level of >750 mg/dl will be additionally examined for at least one of the following parameters to confirm the diagnosis of Fredrickson dyslipidemia Type I, fCs, LPLD:

(1) homozygous or compound heterozygous loss-of-function mutations in genes such as LPL (e.g., P207L, g 188L, D9N), ApoC2, GPIHBP1, ApoC5, or LMF1 that are known to cause Fredrickson dyslipidemia Type I, FCS, LPLD;

(2) LPL activity <20% of normal; and

(3) anti-LPL antibodies.

For each patient diagnosed with Fredrickson dyslipidemia type I, FCS, LPLD, the participation period consists of an examination period of <8 weeks (including an initial 4-week adjusted diet control qualification period), a 1-week study qualification/baseline assessment, a 13-week treatment period, and a 13-week post-treatment assessment period, for a total of 35 weeks of study participation. Patients with a controlled dietary TG level of >440 mg/dL are included in the study. Concomitant medications and adverse events (AEs) are recorded at all study periods.

Patients are placed on a strictly controlled diet (after examination procedures are performed) for the duration of study participation. After 28 days on the controlled diet, patients have baseline measurements and are evaluated for qualification for enrollment in the treatment phase of the study.

Endpoints to be evaluated include: the pharmacodynamic (PD) effects of ISIS 304801 measured by fasting lipoprotein, total ApoC-III, TG, ApoC-II (total and associated with VLDL), apolipoprotein B-100 (apoB-100 and/or or apoB-48), apolipoprotein A-1 (apoA-1), apolipoprotein A-2 (apoA-2), apolipoprotein E (apoE), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), LDL-TG, VLDL-C, VLDL-TG, non-high-density lipoprotein cholesterol (non-HDL-C), non-HDL-TG, HDL-C, HDL-TG, chylomicron cholesterol (CM-C), chylomicron-triglyceride (CM-TG), free fatty acids (FFA) and glycerol levels; the characteristics and postprandial kinetics of lipids, apolipoproteins and lipoproteins and postprandial glucose levels; and, the safety, tolerability and pharmacokinetics (PK) of ISIS 304801. Additional endpoints to evaluate may include a decrease in CETP or an increase in ApoAl, PON1, fat clearance and triglyceride elimination, and an improvement in the ratio of HDL to TG.

Study of Drugs and Treatment

A solution of Study Drug ISIS 304801 (200 mg/ml, 1.0 ml) is provided contained in 2 ml stoppered glass vials. The vials are for single use only. The ISIS 304801 solution and placebo are prepared by a pharmacist (or a qualified delegate). A trained professional administers 300 mg of Study Drug as a single SC injection into the abdomen, thigh, or outer upper arm area on each day of dosing.

Patients receive 13 doses of Study Drug administered by SC injection once a week for 13 weeks (Days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85). Patients complete treatment visits on Day 1 ± 0 days and on Days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 within ± 1 day. Patients in an extensive PK group also visit the clinic on Day 2 and 86 ± 0 days relative to Day 1 and 85, respectively, for a 24-hour blood draw. Patients complete follow-up visits on Day 92 and 99 within ±1 day, Day 127 within ±3 days, and Day 176 within ±5 days of the scheduled visit date. Patients in the postprandial assessment group also visit the clinic on Day 103 within ±2 days and the day following the Day 103 visit for the 24-hour blood draw.

Before each visit that includes a blood draw for PD measurements (Days 8, 15, 29, 43, 57, 71, and 85), patients are provided with a standardized precooked meal for dinner the night before their visit ( to ensure the same moderation of fat intake, per patient and per time point) after which they remain fasted. Alcohol consumption is not permitted during the 48 hours prior to these clinic visits.

Blood is collected after fasting and/or after a meal for measurement of VLDL, ApoC-III and other PD markers on Days 8, 15, 29, 43, 57, 71 and 85 (before administration of the Drug of the Study).

Patients in the postprandial assessment group consume standardized pre-cooked meals (lunches and dinners (provided) and instructions for breakfasts and snacks) for 2 days prior to postprandial assessments. On each of the postprandial assessment days, following blood draws, patients consume a standardized liquid meal, representing approximately one-third of daily caloric requirements, with a stable radioisotope tracer, followed by serial blood sampling. . Patients receive a standardized precooked meal 9 hours after consuming the liquid meal, after which they fast until the 24-hour blood draw the next day.

In addition to minimal sample collection, patients in the extensive PK evaluation group undergo serial blood sampling for 24 hours after their first (Day 1-2) and last (Day 85-86) dose of the Drug. of the Study. PK parameters such as area under the curve (AUC), minimum concentration (Cmin) and others will be evaluated.

Post-treatment Evaluation Period

Patients are followed until Study Day 176. During this time, patients return to the study center for outpatient clinic visits on Study Days 92, 99, 127, and 176 (and Day 103 for patients in the study group). postprandial evaluation) for safety and clinical laboratory evaluations (blood draws), dietary counseling and monitoring, recording of concomitant medication use, and collection of AE events.

Blood samples for PK and PD analysis are collected periodically during the post-treatment evaluation period. Laboratory measurements of serum chemistry, urinalysis, coagulation, complement, hematology, immune function, thyroid function, and complete lipid panel are performed at different times throughout the study.

Postprandial assessments are performed in a subset of patients as described below.

Post-prandial Meal, Sampling and Evaluation Program

Postprandial assessment for lipoprotein metabolism is performed using a radiolabeled meal supplemented with a labeled tracer, 3H-palmitate (300 jCi, Perkin Elmer Inc., Woodbridge, ON, Canada), sonicated into the liquid meal. Palmitate is a fatty acid that is a common component of any diet. The 3H-palmitate tracer emits weak radioactivity, equivalent to an the circulation. The methodology to be applied to study the postprandial kinetics of the appearance and clearance of chylomicrons is well established (Mittendorfer et al. 2003, Diabetes, 52: 1641-1648; Bickerton et al. 2007; Normand-Lauziere et al. 2010, PLoS. One, 5: e10956).

A liquid meal (similar to a shake) containing a small amount (300|<j>C¡) of radiolabeled fatty acids (3H-palmitate) will be provided. Liquid food will provide approximately one-third of your daily calorie requirements. From 1 hr before to 9 hrs after meal ingestion, a constant infusion of [U-13C]-K palmitate (0.01 jmol/kg/min in 100 ml of 25% human serum albumin; Cambridge Isotopes Laboratories Inc., Andover, MA) and a continuous infusion (0.05 pmol/kg/min) prepared (1.6 pmol/kg) of [1,1,2,3,3-2H]-glycerol Cambridge Isotopes Laboratories Inc.) as described previously (Normand-Lauziere et al. 2010, PLoS. One, 5: e10956). The rates of appearance of palmitate and glycerol in plasma are calculated using Steele's unsteady state equation assuming a volume of distribution of 90 ml/kg and 230 ml/kg, respectively (Gastaldelli et al. 1999, J Appl. Physiol, 87: 1813-1822).

Blood samples are drawn at intervals before and after ingestion of the radiolabeled meal on the days before and after the Treatment phase, as indicated in the table below. Participants are given a standardized meal after the 9-hour blood draw. Blood is collected in tubes containing Na2 EDTA and Orlistat (30 jg/ml, Roche, Mississauga, Canada) to avoid triacylglycerol lipolysis in vitro and separate samples will be collected in NaF tubes for plasma glucose determination.

The following is measured in the time point layer:

• Plasma fraction and CM levels for 3H tracer

• Appearance rates of [U-13C]-K palmitate and [1,1,2,3,3-2H]-glycerol in plasma

• Plasma fraction and CM levels for TG, TC and apoB

• Plasma fraction and VLDL levels for apo CIII, apo CII and apo E

• Plasma levels for glucose

Plasma samples may also be used for profiling of drug-binding proteins, bioanalytical method validation purposes, stability and metabolite assessments, or to evaluate other actions of ISIS 304801 with plasma constituents.

Results

The results are presented below for three patients diagnosed with Fredrickson dyslipidemia Type I, FCS, LPLD, recruited for this study. Two patients are homozygous for the P207L null LPL gene mutation and one patient is compound heterozygous for the P207L and G188E null LPL gene mutations. All patients have LPL mass but no or extremely low levels (<5%) of LPL activity. Patients had a TG level >440 mg/dl after the diet but before starting treatment. Two of the patients had a confirmed history of acute pancreatitis and one had received gene therapy with GlyberaR in December 2007.

Data for the percentage change in fasting ApoCIII levels are presented in the Table below. The results indicate that treatment with ISIS 304801 reduced fasting levels of ApoC-III. 'n.d.' indicates that data had not yet been collected for that particular time point.

Table 1

Fasting triglyceride levels were also measured. Data for the percentage change, as well as absolute levels, of fasting triglyceride levels are presented in the Tables below. The results indicate that treatment with ISIS 304801 reduced fasting triglyceride levels.

Table 2

Table 3

Fasting non-HDL cholesterol levels were also measured. Data for the percentage change, as well as absolute levels, of fasting non-HDL cholesterol levels are presented in the Tables below. The results indicate that treatment with ISIS 304801 reduced fasting levels of non-HDL cholesterol.

Table 4

Table 5

ApoB-48 levels, a measure of chylomicrons, were also measured. Data for the percentage change, as well as absolute levels, of ApoB-48 levels are presented in the Tables below. The results indicate that treatment with ISIS 304801 reduced fasting levels of ApoB-48.

Table 6

Table 7

The global lipid profile in patients with fasting FCS was measured at the end of treatment and compared with the reference value. The data are presented in the Tables below and indicate that treatment with ISIS 304801 improved the overall lipid profile in the patients.

Table 8

Table 9

Safety assessment

Treatment with ISIS 304801 did not have any problems with elevations in liver enzymes more than three times the ULN, abnormalities in renal function, significant clinical changes in other laboratory values, or related to significant SAEs or AEs.

Treatment was tolerated by all patients without flu-like symptoms and infrequent mild site reactions, which resolved without treatment. There were no interruptions due to injection site reactions.

Claims (27)

1. A specific inhibitor of ApoCIII for use in a method to i) treat, prevent, delay or ameliorate lipoprotein lipase deficiency (LPLD) or familial chylomicronemia syndrome (FCS) in an animal; or ii) prevent, delay or ameliorate pancreatitis, or symptoms thereof in an animal with LPLD or FCS, wherein the specific ApoCIII inhibitor comprises an antisense compound targeting ApoCIII, wherein the antisense compound is an RNA interfering compound . 2. The specific ApoCIII inhibitor for use according to claim 1, wherein the interfering RNA compound is single-stranded. 3. The specific ApoCIII inhibitor for use according to claim 1, wherein the interfering RNA compound is double stranded. 4. The specific ApoCIII inhibitor for use according to any preceding claim, wherein the interfering RNA compound is, or is at least, 80%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a nucleobase sequence of an ApoCIII nucleic acid. 5. The specific ApoCIII inhibitor for use according to claim 4, wherein the interfering RNA compound is complementary to a nucleobase sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO : 4. 6. The specific ApoCIII inhibitor for use according to any preceding claim, wherein the interfering RNA compound comprises at least one modified nucleoside comprising a modified sugar moiety or at least one modified tetrahydropyran nucleoside. 7. The specific ApoCIII inhibitor for use according to any of claims 1-5, wherein the interfering<a>R<n>compound comprises at least one modified nucleoside comprising a modified sugar moiety comprising a substituent group selected from 2'-F and 2'-OMe, or comprises a bridge between the 4' and 2' ribosyl ring atoms, wherein the bridge is selected from LNA and cEt. 8. The specific ApoCIII inhibitor for use according to any of claims 6 and 7, wherein the interfering RNA compound comprises a region in which each nucleoside is a 2'-F nucleoside, or a region in which each nucleoside is a 2'-OMe nucleoside. 9. The specific ApoCIII inhibitor for use according to any preceding claim, wherein the interfering RNA compound comprises one or more regions of alternating sugar modifications; optionally wherein the alternate modifications are 2'-F and 2'-OMe. 10. The specific ApoCIII inhibitor for use according to any preceding claim, wherein at least one nucleoside of the interfering RNA compound comprises a modified nucleobase. 11. Specific ApoCIII inhibitor for use according to claim 10, wherein at least one nucleobase of the interfering RNA compound is hypoxanthine. 12. The specific ApoCIII inhibitor for use according to any preceding claim, wherein at least one internucleoside bond of the interfering RNA compound is a modified internucleoside bond. 13. The specific ApoCIII inhibitor for use according to claim 12, wherein at least one internucleoside bond of the interfering RNA compound is a phosphorothioate internucleoside bond. 14. The specific ApoCIII inhibitor for use according to claim 12, wherein each internucleoside bond of the interfering RNA compound is selected from a phosphodiester internucleoside bond and a phosphorothioate internucleoside bond. 15. The specific ApoCIII inhibitor for use according to claim 3, wherein the double-stranded interfering RNA compound has one or more single-stranded regions or overhangs. 16. The specific ApoCIII inhibitor for use according to any preceding claim, wherein the interfering RNA compound comprises a modified 5'-terminal end. 17. Specific ApoCIII inhibitor for use according to claim 16, wherein the 5'-terminal end comprises a modified phosphate moiety. 18. The specific ApoCIII inhibitor for use according to any preceding claim, wherein the interfering RNA compound comprises inverted deoxyabase caps attached to one or both ends of the interfering RNA compound. 19. The specific ApoCIII inhibitor for use according to any preceding claim, wherein the antisense compound is a conjugated antisense compound, optionally wherein the antisense compound is conjugated to a conjugated carbohydrate group. 20. The specific ApoCIII inhibitor for use according to claim 2, wherein the single-stranded interfering RNA compound comprises: (a) a modified 5'-terminal end, optionally comprising a modified phosphate moiety; or (b) one or more types of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif, optionally wherein the oligonucleotide comprises a region (i) having uniform sugar modifications or (ii) alternating sugar modifications; either (c) modified internucleoside linkages arranged along an oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif, optionally wherein the oligonucleotide comprises a region (i) having an alternating internucleoside linkage motif or (ii) ) of uniformly modified internucleoside bonds. 21. A pharmaceutical composition comprising the specific ApoCIII inhibitor as defined in any preceding claim and a pharmaceutically acceptable carrier or diluent for use in a method for i) treating, preventing, delaying or ameliorating LPLD or FCS in an animal, or ii) prevent, delay or improve pancreatitis, or symptoms thereof in an animal with LPLD or FCS. 22. The specific ApoCIII inhibitor for use according to any of claims 1 to 20, or the pharmaceutical composition for use according to claim 21, wherein: (a) the specific ApoCIII inhibitor or pharmaceutical composition is for administration parenterally, optionally subcutaneously; (b) the specific ApoCIII inhibitor or the pharmaceutical composition is for administration in combination with a second agent, optionally wherein the second agent is selected from ApoCIII lowering agent, cholesterol lowering agent, non-HDL lipid lowering agent, agent LDL lowering agent, TG lowering agent, cholesterol lowering agent, HDL raising agent, fish oil, niacin, fibrate, statin, DCCR (diazoxide salt), glucose lowering agent or antidiabetic agent, and wherein the second agent optionally it is for administration concomitantly or sequentially with the specific ApoCIII inhibitor or the pharmaceutical composition; I (c) the specific ApoCIII inhibitor is in salt form. 23. The specific ApoCIII inhibitor or the pharmaceutical composition for use according to any of claims 1 to 21, wherein the specific ApoCIII inhibitor or the pharmaceutical composition decreases fasting triglyceride levels by at least 40% with respect to the reference value in the animal. 24. The specific ApoCIII inhibitor or the pharmaceutical composition for use according to claim 23, wherein the specific ApoCIII inhibitor or the pharmaceutical composition decreases fasting triglyceride levels by at least 45%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% in the animal. 25. The specific ApoCIII inhibitor or pharmaceutical composition for use according to claim 24, wherein the specific ApoCIII inhibitor or pharmaceutical composition decreases fasting triglyceride levels by at least 70% in the animal. 26. The specific ApoCIII inhibitor or the pharmaceutical composition for use according to any of claims 1 to 21, wherein the specific ApoCIII inhibitor or the pharmaceutical composition decreases fasting triglyceride levels to <750 mg/dl, <700 mg/dl, <650 mg/dl, < 600 mg/dl, <550 mg/dl, <500 mg/dl, <450 mg/dl, <400 mg/dl, <350 mg/dl, <300 mg/dl, <250 mg/dl, <200 mg/dl <150 mg/dl or <100 mg/dl in the animal. 27. The specific ApoCIII inhibitor or pharmaceutical composition for use according to claim 26, wherein the specific ApoCIII inhibitor or pharmaceutical composition decreases fasting triglyceride levels to <400 mg/dl in the animal.